Insecticidal proteins and methods of use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are isolated insecticidal proteins and nucleic acids. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling or killing lepidopteran, coleopteran, dipteran, fungal, hemipteran, and nematode pest populations and for producing compositions with insecticidal activity.

This application is a continuation of U.S. Ser. No. 14/775,344 filedSep. 11, 2015, now U.S. Pat. No. 9,879,277, which is 371 (NationalStage) of PCT/US14/25286 filed Mar. 13, 2014, which claims the benefitof U.S. application Ser. No. 13/803,634 filed Mar. 14, 2013, which areincorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically asan ASCII formatted sequence listing with a file named“5264WOPCT_Sequence_Listing.TXT” and is filed concurrently with thespecification. The sequence listing contained in this ASCII formatteddocument is part of the specification and is herein incorporated byreference in its entirety.

FIELD

The present disclosure relates to genes that encode pesticidalpolypeptides characterized by pesticidal activity against insect pests.Compositions and methods of the disclosure utilize the disclosed nucleicacids and their encoded pesticidal polypeptides to control plant pests.

BACKGROUND

Insect pests are a major factor in the loss of the world's agriculturalcrops. For example, armyworm feeding, black cutworm damage, or Europeancorn borer damage can be economically devastating to agriculturalproducers. Insect pest-related crop loss from European corn borerattacks on field and sweet corn alone has reached about one billiondollars a year in damage and control expenses.

Traditionally, the primary method for controlling insect pestpopulations is the application of broad-spectrum chemical insecticides.However, consumers and government regulators alike are becomingincreasingly concerned with the environmental hazards associated withthe production and use of synthetic chemical pesticides. Because of suchconcerns, regulators have banned or limited the use of some of the morehazardous pesticides. Thus, there is substantial interest in developingalternative pesticides.

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria, or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards, and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a broad range of insect pestsincluding Lepidoptera, Diptera, Coleoptera, Hemiptera, and others.Bacillus thuringiensis (Bt) and Bacillus papilliae are among the mostsuccessful biocontrol agents discovered to date. Insect pathogenicityhas also been attributed to strains of B. larvae, B. lentimorbus, B.sphaericus and B. cereus. Microbial insecticides, particularly thoseobtained from Bacillus strains, have played an important role inagriculture as alternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. These genetically engineered crops are now widely used inAmerican agriculture and have provided producers with an environmentallyfriendly alternative to traditional insect-control methods. While theyhave proven to be very successful commercially, these geneticallyengineered, insect-resistant crop plants typically provide resistance toonly a narrow range of economically important pests. Some insects havedeveloped resistance to some insecticidal polypeptides in thesegenetically engineered crops.

Accordingly, there remains a need for new pesticidal proteins with abroader range of insecticidal activity against insect pests, e.g.,toxins which are active against a greater variety of insects from theorder Lepidoptera, Coleoptera, Hemiptera, and others. In addition, thereremains a need for biopesticides having improved insecticidal activity,and activity against insects that have developed resistance to existingpesticides and pesticidal proteins.

SUMMARY

In one aspect compositions and methods for conferring pesticidalactivity to bacteria, plants, plant cells, tissues and seeds areprovided. Compositions include nucleic acid molecules encoding sequencesfor pesticidal and insecticidal polypeptides, vectors comprising thosenucleic acid molecules, and host cells comprising the vectors.Compositions also include the pesticidal polypeptide sequences andantibodies to those polypeptides. The nucleic acid sequences can be usedin DNA constructs or expression cassettes for transformation andexpression in organisms, including microorganisms and plants. Thenucleotide or amino acid sequences may be synthetic sequences that havebeen designed for expression in an organism including, but not limitedto, a microorganism or a plant. Compositions also comprise transformedbacteria, plants, plant cells, tissues and seeds.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding MP467 or MP812 polypeptides, including amino acidsubstitutions, deletions, insertions, fragments of SEQ ID NO: 2 or SEQID NO: 24. Additionally, amino acid sequences corresponding to the MP467or MP812 polypeptides are encompassed. Provided are isolated orrecombinant nucleic acid molecules of SEQ ID NO: 1 or SEQ ID NO: 23capable of encoding MP467 or MP812 polypeptides as well as amino acidsubstitutions, deletions, insertions, fragments thereof, andcombinations thereof. Nucleic acid sequences that are complementary to anucleic acid sequence of the embodiments or that hybridize to a sequenceof the embodiments are also encompassed.

In another aspect methods are provided for producing the polypeptidesand for using those polypeptides for controlling or killing aLepidopteran, Coleopteran, nematode, fungi, and/or Hemipteran pests. Thetransgenic plants of the embodiments express one or more of thepesticidal sequences disclosed herein. In various embodiments, thetransgenic plant further comprises one or more additional genes forinsect resistance, for example, one or more additional genes forcontrolling Coleopteran, Lepidopteran, Hemipteran or nematode pests. Itwill be understood by one of skill in the art that the transgenic plantmay comprise any gene imparting an agronomic trait of interest.

In another aspect methods for detecting the nucleic acids andpolypeptides of the embodiments in a sample are also included. A kit fordetecting the presence of a MP467 polypeptide or a MP812 polypeptide ordetecting the presence of a polynucleotide encoding a MP467 polypeptideand/or a MP812 polypeptide in a sample is provided. The kit may beprovided along with all reagents and control samples necessary forcarrying out a method for detecting the intended agent, as well asinstructions for use.

In another aspect the compositions and methods of the embodiments areuseful for the production of organisms with enhanced pest resistance ortolerance. These organisms and compositions comprising the organisms aredesirable for agricultural purposes. The compositions of the embodimentsare also useful for generating altered or improved proteins that havepesticidal activity or for detecting the presence of MP467 polypeptidesor nucleic acids and MP812 polypeptides or nucleic acids in products ororganisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an amino acid sequence alignment of MP467 (SEQ ID NO: 2)with a hydralysin protein (SEQ ID NO: 4) from Hydra viridissima.Identical and similar amino acids between MP467 (SEQ ID NO: 2) andhydralysin (SEQ ID NO: 4) are highlighted (

=identical;

=similar). The residues (Phe26, Val48, Pro50, Ile52, Tyr56, Met193,Val202, His205, Tyr206, Phe207, Trp208, Phe209, and Leu210) constitutingthe hydrophobic patch of MP467 (SEQ ID NO: 2) are indicated by a “*”below the sequence.

FIG. 2 shows an amino acid sequence alignment of MP467 (SEQ ID NO: 2)with a parasporin 2/Cry46Ab protein (SEQ ID NO: 12) from Bacillusthuringiensis. Identical and similar amino acids between MP467 (SEQ IDNO: 2) and Cry46Ab (SEQ ID NO: 12) are highlighted (

=identical;

=similar). The residues (Phe26, Val48, Pro50, Ile52, Tyr56, Met193,Val202, His205, Tyr206, Phe207, Trp208, Phe209, and Leu210) constitutingthe hydrophobic patch of MP467 (SEQ ID NO: 2) are indicated by a “*”below the sequence.

FIG. 3 shows an amino acid sequence alignment of MP467 (SEQ ID NO: 2)with a parasporin 2/Cry46Aa protein (SEQ ID NO: 10) from Bacillusthuringiensis. Identical and similar amino acids between MP467 (SEQ IDNO: 2) and Cry46Aa (SEQ ID NO: 10) are highlighted (

=identical;

=similar). The residues (Phe26, Val48, Pro50, Ile52, Tyr56, Met193,Val202, His205, Tyr206, Phe207, Trp208, Phe209, and Leu210) constitutingthe hydrophobic patch of MP467 (SEQ ID NO: 2) are indicated by a “*”below the sequence.

FIG. 4 shows an amino acid sequence alignment of MP467 (SEQ ID NO: 2)with the insecticidal inactive homologs MP543 (SEQ ID NO: 6) and MP544(SEQ ID NO: 8). The sequence diversity between MP543 (SEQ ID NO: 6) andMP544 (SEQ ID NO: 8) compared to MP467 (SEQ ID NO: 2) is highlighted Theresidues (Phe26, Val48, Pro50, Ile52, Tyr56, Met193, Val202, His205,Tyr206, Phe207, Trp208, Phe209, and Leu210) constituting the hydrophobicpatch of MP467 (SEQ ID NO: 2) are indicated by a “*” below the sequence.The alternating residues in the β hairpins are underlined and indicatedby a “+” below the sequence.

FIG. 5 shows an amino acid sequence alignment of Beta hairpin structureregions from MP467 (467) (SEQ ID NO: 14) and homologs: parasporin-2(Ps2) (SEQ ID NO: 15); hydralysin (Hdr) (SEQ ID NO: 16); alpha toxin(ApT) (SEQ ID NO: 17); aerolysin (Aer) (SEQ ID NO: 18); ε-toxin (Epn)(SEQ ID NO: 19); hemolytic lectin (LSL) (SEQ ID NO: 20); enterotoxin(CPE) (SEQ ID NO: 21); and alpha-hemolysin (Aph) (SEQ ID NO: 22). Thealternating residues in the β hairpins are highlighted.

FIG. 6 shows the structure of the pore stem of α-hemolysin indicatingthe β-hairpin region.

FIG. 7 shows the overall three-domain structure of MP467, MP812, and Cry46A.

FIG. 8 shows structural comparisons between MP467 homologs.

FIG. 9 shows the structure of Domain 1 of MP467 and MP812 indicatinghelix 1 (H1), helix 2 (H2), helix 3 (H3), helix 4 (H4). β strand 3 (S3),β strand 11 (S11), β strand 12 (S12), and the 1′-β-turn region between βstrand 11 and β strand 12. Residues H205, Y206, F207, W208, and F209 ofthe 1′-β-turn region of MP467 (SEQ ID NO: 2) are highlighted. Thecorresponding residues H229, H230, F231, W232, and A233 of MP812 (SEQ IDNO: 24) are boxed.

FIG. 10 shows an amino acid sequence alignment of MP467 (SEQ ID NO: 2)and MP812 (SEQ ID NO: 24. Identical and similar amino acids betweenMP467 (SEQ ID NO: 2) and MP812 (SEQ ID NO: 12) are highlighted (

=identical;

=similar).

DETAILED DESCRIPTION

The embodiments of the disclosure are drawn to compositions and methodsfor controlling insect pests, particularly plant pests. Morespecifically, the isolated nucleic acid of the embodiments, andfragments and variants thereof, comprise nucleotide sequences thatencode pesticidal polypeptides (e.g., proteins). The disclosedpesticidal proteins are biologically active (e.g., pesticidal) againstinsect pests such as, but not limited to, insect pests of the orderLepidoptera, Coleoptera, and Hemiptera. Insect pests of interestinclude, but are not limited to: Ostrinia nubilalis (European CornBorer), Spodoptera frugiperda (Fall Armyworm), Helicoverpa zea Boddie(Corn Earworm), Agrotis ipsilon Hufnagel (Black Cutworm), Pseudoplusiaincludens Walker (Soybean Looper), Anticarsia gemmatalis Hübner(Velvetbean Caterpillar), Diabrotica virgifera virgifera (Western CornRootworm), Southern Corn Rootworm (Diabrotica spp.), Northern CornRootworm (Diabrotica spp.), Mexican Bean Beetle (Epilachna varivestisMulsant), Stinkbugs (family Pentatomidae) and Lygus spp.

The compositions of the embodiments comprise isolated nucleic acids, andfragments and variants thereof that encode pesticidal polypeptides,expression cassettes comprising nucleotide sequences of the embodiments,isolated pesticidal proteins and variants and fragments thereof, andpesticidal compositions.

The following embodiments are encompassed by the present disclosure:

1. An isolated three-domain insecticidal protein having a structurecomprising:

-   -   a. a Domain I, comprising a surface hydrophobic patch and a type        1′ β-turn;    -   b. a Domain II; and    -   c. a Domain III, wherein the surface of Domain II and Domain III        comprise a stripe of solvent exposed serine and threonine        residues.        2. The isolated three-domain insecticidal protein of embodiment        1, wherein Domain I further comprises an anti-parallel β-sheet        with four short strands and four α-helices designated as helix        1, helix 2, helix 3, and helix 4.        3. The isolated three-domain insecticidal protein of embodiment        2, wherein the anti-parallel β-sheet of Domain I further        comprises the C-terminal end of strand 11, strand 12, and the        type 1′ β-turn joins strand 11 and strand 12.        4. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 3, wherein Domain I further comprises a        β-hairpin between helix 2 and helix 3.        5. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 4, wherein the surface hydrophobic patch        comprises the residues from strand 12, helix 2, helix 3, and the        type 1′ β-turn comprises the residues between strand 11 and        strand 12.        6. The isolated three domain insecticidal protein of embodiment        5, wherein the type 1′ β-turn between strand 11 and strand 12        comprises an amino acid sequence motif as represented by SEQ ID        NO: 25.        7. The isolated three domain insecticidal protein of embodiment        5 or 6, wherein the type 1′ β-turn between strand 11 and strand        12 comprises the amino acid sequence of SEQ ID NO: 13.        8. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 7 wherein the surface hydrophobic patch is        about 180-220 Å².        9. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 8 wherein the hydrophobic patch comprises        residues corresponding to Phe26, Val48, Pro50, Ile52, Tyr56,        Met193, Val202, His205, Tyr206, Phe207, Trp208, Phe209, and        Leu210 of SEQ ID NO: 2.        10. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 9, wherein Domain 1 comprises residue 1 to        about residue 65 and about residue 187 to about residue 223        corresponding to the residues of SEQ ID NO: 2.        11. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 10, wherein Domain II comprises five-stranded        anti-parallel β-sheet (β5/β6-β11-β13-β7-β10) patched on one side        by an amphipathic β-hairpin stemmed from β2 and β10.        12. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 11, wherein Domain II comprises about residue        66 to about residue 79; about residue 104 to about residue 154;        about residue 175 to about residue 186; and about residue 224 to        about residue 234 corresponding to the residues of SEQ ID NO: 2.        13. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 12, wherein Domain III comprises the same five        β-strands of Domain II which extend and refold into domain III        with a beta-sandwich structure, wherein the three strands β5/β6,        β11, and β13 make a 180° twist in the middle forming a new 3        stranded β-sheet as one side of a β-sandwich and β7 and β10        spray from the central sheet, twist in middle, and        hydrophobically pack against strands β5/β6, β11, and β13.        14. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 13, wherein Domain III comprises from about        residue 80 to about residue 103; about residue 155 to about        residue 174; and about residue 235 to about residue 246        corresponding to the residues of SEQ ID NO: 2.        15. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 14, wherein the stripe of exposed serine and        threonine residues on the surface of Domain II and Domain III        comprises: 3 serine and 4 threonine residues in an alternating        motif on strand 11, and 3 serine and 2 threonine residues on        strand 7.        16. The isolated three-domain insecticidal protein of embodiment        15, wherein the 3 serine and 4 threonine residues on strand 11        have an average solvent accessibility of about 78.5 Å² and the 3        serine and 2 threonine residues on strand 7 average solvent        exposure of about 59.3 Å².        17. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 16, wherein the three-domain insecticidal        protein has reduced hemolytic activity compared to the        polypeptide of SEQ ID NO: 2.        18. The isolated three-domain insecticidal protein of embodiment        17, wherein the hemolytic activity is reduced at least 2 fold        compared to the hemolytic activity of the polypeptide of SEQ ID        NO: 2.        19. The isolated three-domain insecticidal protein of embodiment        17, wherein the hemolytic activity is reduced greater than 20        fold compared to the hemolytic activity of the polypeptide of        SEQ ID NO: 2.        20. The isolated three-domain insecticidal protein of any one of        embodiments 17 to 19, wherein the reduction in hemolytic        activity is the result of one or more amino acid substitutions        at a residue in the hydrophobic patch.        21. The isolated three-domain insecticidal protein of embodiment        20, wherein the residue in the hydrophobic patch is selected        from a residue corresponding to Phe26, Val48, Pro50, Ile52,        Tyr56, Met193, Val202, His205, Tyr206, Phe207, Trp208, Phe209,        and Leu210 of SEQ ID NO: 2.        22. The isolated three-domain insecticidal protein of embodiment        21, wherein the residue in the hydrophobic patch is selected        from a residue corresponding to Tyr56, Tyr206, Phe207, Trp208,        Phe209 of SEQ ID NO: 2.        23. The isolated three-domain insecticidal protein of embodiment        20, wherein the hydrophobic patch comprises a motif as        represented by a sequence of SEQ ID NO: 26, wherein at least one        residue is an amino acid different from the wild type amino acid        at the residue corresponding to position 206, 207 208 or 209 of        SEQ ID NO: 2.        24. The isolated three-domain insecticidal protein of embodiment        20, wherein the hydrophobic patch comprises a motif as        represented by a sequence of SEQ ID NO: 27, wherein at least one        residue is an amino acid different from the wild type amino acid        at the residue corresponding to position 206, 207 208 or 209 of        SEQ ID NO: 2.        25. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 24, wherein the three-domain insecticidal        protein has at least 80% identity to SEQ ID NO: 2.        26. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 24, wherein the three-domain insecticidal        protein has at least 90% identity to SEQ ID NO: 2.        27. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 24, wherein the three-domain insecticidal        protein has at least 95% identity to SEQ ID NO: 2.        28. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 24, wherein the three-domain insecticidal        protein has at least 80% identity to SEQ ID NO: 24.        29. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 24, wherein the three-domain insecticidal        protein has at least 90% identity to SEQ ID NO: 24.        30. The isolated three-domain insecticidal protein of any one of        embodiments 1 to 24, wherein the three-domain insecticidal        protein has at least 95% identity to SEQ ID NO: 24.        31. An isolated nucleic acid molecule encoding the three-domain        insecticidal protein of any one of embodiments 1 to 30.        32. The isolated nucleic acid molecule of embodiment 31, wherein        said nucleic acid molecule is a synthetic molecule that has been        designed for expression in a plant.        33. A DNA construct comprising the nucleic acid molecule of        embodiment 31 or 32.        34. The DNA construct of embodiment 33, wherein the DNA        construct further comprises a heterologous promoter operably        linked to the nucleic acid molecule encoding the three-domain        insecticidal protein.        35. A host cell comprising the DNA construct of embodiment 33 or        34.        36. The host cell of embodiment 35, wherein the host cell is a        bacterial cell.        37. The host cell of embodiment 35, wherein the host cell is a        plant cell.        38. A transgenic plant comprising the DNA construct of        embodiment 33 or 34.        39. The transgenic plant of embodiment 38, wherein the plant is        selected from the group consisting of: maize, sorghum, wheat,        sunflower, tomato, cruciferous species, capsicum species,        potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco,        barley, and oilseed rape.        40. Transformed seed of the transgenic plant of embodiment 38 or        39, wherein the seed comprise the DNA construct.        41. A composition comprising the three-domain insecticidal        protein of any one of embodiments 1 to 30.        42. The composition of embodiment 41, wherein said composition        is selected from the group consisting of a powder, dust, pellet,        granule, spray, emulsion, colloid, and solution.        43. The composition of embodiment 42, wherein said composition        is prepared by desiccation, lyophilization, homogenization,        extraction, filtration, centrifugation, sedimentation, or        concentration of a culture of micro-organisms.        44. A method for controlling a pest population comprising        contacting said population with an insecticidally-effective        amount of the three-domain insecticidal protein of any one of        embodiments 1 to 30.        45. A method for killing a pest comprising contacting said pest        with, or feeding to said pest, an insecticidally-effective        amount of the three-domain insecticidal protein of any one of        embodiment 1 to 30.        46. A method for protecting a plant from a pest, comprising        introducing into said plant or cell thereof at least one DNA        construct of embodiment 33 or 34.        47. The method of embodiment 46, wherein the plant produces an        insecticidal protein having insecticidal activity against at        least one insect species in the order Hemiptera.        48. The method of embodiment 47, wherein the insect species is        in the family Pentatomidea.        49. The method of embodiment 48, wherein the insect species is        selected from Brown Marmorated Stink Bug (Halyomorpha halys),        Southern green stink bug (Nezara viridula), green stink bug        (Chinavia hilare), Brown stink bug (Euschistus servus), Dusky        stink bug (Euschistus tristigmus), Euschistus quadrator, Rice        stink bug (Oebalus pugnax), Redshouldered stink bug (Thyanta        accerra McAtee), Thyanta custator, Redbanded stink bug        (Piezodorus guildini), Harlequin bug (Murgantia histrionica),        Edessa bifida, and Twice-stabbed stink bug (Cosmopepla        lintneriana Kirkaldy).        50. The method of embodiment 46, wherein the plant produces an        insecticidal protein having insecticidal activity against at        least one insect species in the order Lepidoptera.        51. The method of embodiment 50, wherein the insect species is        selected from European corn borer (Ostrinia nubilalis), corn        earworm (Helicoverpa zea), black cutworm (Agrotis ipsilon), fall        armyworm (Spodoptera frugiperda), Soybean looper (Pseudoplusia        includens) and Velvet bean caterpillar (Anticarsia gemmatalis).        52. The method of embodiment 46, wherein the plant produces an        insecticidal protein having insecticidal activity against at        least one Coleoptera species.        53. The method of embodiment 52, wherein the insect species is        selected from Western corn rootworm (Diabrotica virgifera        virgifera), Northern corn rootworm (Diabrotica barbers), Mexican        corn rootworm (Diabrotica virgifera zeae).        54. A method for producing a three-domain insecticidal        polypeptide, comprising culturing the host cell of any one of        embodiments 35 to 37 under conditions in which the nucleic acid        molecule encoding the polypeptide is expressed.        55. A plant having stably incorporated into its genome a DNA        construct comprising a nucleotide sequence that encodes a        protein having at least 80% sequence identity to an amino acid        sequence of SEQ ID NO: 10; wherein said nucleotide sequence is        operably linked to a promoter that drives expression of the        coding sequence in a plant cell.        56. A method for protecting a plant from a pest, comprising        introducing into said plant or cell thereof at least one DNA        construct comprising a nucleotide sequence that encodes a        insecticidal polypeptide, having at least 80% sequence identity        to the amino acid sequence of SEQ ID NO: 10.        57. The method of embodiment 56, wherein the plant produces an        insecticidal protein having insecticidal activity against at        least one insect species in the order Hemiptera.        58. The method of embodiment 57, wherein the insect species is        in the family Pentatomidea.

The embodiments further provide isolated pesticidal (e.g., insecticidal)polypeptides encoded by either a naturally-occurring or modified nucleicacid of the embodiments. More specifically, the embodiments providepolypeptides comprising an amino acid sequence set forth in SEQ ID NO: 2and SEQ ID NO: 24, and the polypeptides encoded by nucleic acidsdescribed herein, for example those set forth in SEQ ID NO: 1 and SEQ IDNO: 23, and fragments and variants thereof, including but not limited tothe polypeptides of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69.

The embodiments further provide isolated pesticidal (e.g., insecticidal)polypeptides More specifically, the embodiments provide polypeptidescomprising an amino acid sequence set forth in SEQ ID NO: 2 and SEQ IDNO: 24, fragments and variants thereof, including but not limited to SEQID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO:38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ IDNO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO:57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ IDNO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69.

In some embodiments the insecticidal polypeptide has at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 24, SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67,SEQ ID NO: 68, or SEQ ID NO: 69.

In some embodiments the insecticidal polypeptide has at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 2.

In some embodiments the insecticidal polypeptide has at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 24.

In some embodiments the insecticidal polypeptide has at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID NO: 10.

Some embodiments provide modified pesticidal polypeptides characterizedby improved insecticidal activity relative to the pesticidal activity ofthe corresponding wild-type protein. The embodiments further provideplants and microorganisms transformed with these novel nucleic acids,and methods involving the use of such nucleic acids, pesticidalcompositions, transformed organisms, and products thereof in controllinginsect pests.

The nucleic acids and nucleotide sequences of the embodiments may beused to transform any organism to produce the encoded pesticidalproteins. Methods are provided that involve the use of such transformedorganisms to control plant pests. The nucleic acids and nucleotidesequences of the embodiments may also be used to transform organellessuch as chloroplasts (McBride et al. (1995) Biotechnology 13: 362-365;and Kota et al. (1999) Proc. Natl. Acad. Sci. USA 96: 1840-1845).

The embodiments further relate to the identification of fragments andvariants of the naturally-occurring coding sequence that encodebiologically active pesticidal proteins. The nucleotide sequences of theembodiments find direct use in methods for controlling pests.Accordingly, the embodiments provide new approaches for controllinginsect pests that do not depend on the use of traditional, syntheticchemical insecticides. The embodiments involve the discovery ofnaturally-occurring, biodegradable pesticides and the genes that encodethem.

The embodiments further provide fragments and variants of the naturallyoccurring coding sequence that also encode biologically active (e.g.,pesticidal) polypeptides. The nucleic acids of the embodiments encompassnucleic acid or nucleotide sequences that have been optimized forexpression by the cells of a particular organism, for example nucleicacid sequences that have been back-translated (i.e., reverse translated)using plant-preferred codons based on the amino acid sequence of apolypeptide having enhanced pesticidal activity. The embodiments furtherprovide mutations which confer improved or altered properties on thepolypeptides of the embodiments. See, e.g., copending U.S. applicationSer. No. 10/606,320, filed Jun. 25, 2003, and Ser. No. 10/746,914, filedDec. 24, 2003.

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe embodiments.

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above terms are more fully defined byreference to the specification as a whole.

By “pesticidal toxin” or “pesticidal protein” is intended a protein thathas toxic activity against one or more pests, including, but not limitedto, members of the Lepidoptera, Diptera, Hemiptera, and Coleopteraorders, or the Nematoda phylum, or a protein that has homology to such aprotein. Pesticidal proteins have been isolated from organismsincluding, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp.,Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogues of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

Polypeptides of the embodiments can be produced either from a nucleicacid disclosed herein, or by the use of standard molecular biologytechniques. For example, a protein of the embodiments can be produced byexpression of a recombinant nucleic acid of the embodiments in anappropriate host cell, or alternatively by a combination of ex vivoprocedures.

As used herein, the terms “isolated” and “purified” are usedinterchangeably to refer to nucleic acids or polypeptides orbiologically active portions thereof that are substantially oressentially free from components that normally accompany or interactwith the nucleic acid or polypeptide as found in its naturally occurringenvironment. Thus, an isolated or purified nucleic acid or polypeptideis substantially free of other cellular material or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

In some embodiments the polypeptides of the disclosure include aminoacid sequences deduced from the full-length nucleic acid sequencesdisclosed herein, and amino acid sequences that are shorter than thefull-length sequences, either due to the use of an alternate downstreamstart site, or due to processing that produces a shorter protein havingpesticidal activity. Processing may occur in the organism the protein isexpressed in, or in the pest after ingestion of the protein.

In some embodiments the nucleic acid molecule encoding the MP467polypeptide or MP812 polypeptide is a non-genomic nucleic acid sequence.As used herein a “non-genomic nucleic acid sequence” or “non-genomicnucleic acid molecule” or “non-genomic polynucleotide” refers to anucleic acid molecule that has one or more change in the nucleic acidsequence compared to a native or genomic nucleic acid sequence. In someembodiments the change to a native or genomic nucleic acid moleculeincludes but is not limited to: changes in the nucleic acid sequence dueto the degeneracy of the genetic code; codon optimization of the nucleicacid sequence for expression in plants; changes in the nucleic acidsequence to introduce at least one amino acid substitution, insertion,deletion and/or addition compared to the native or genomic sequence;removal of one or more intron associated with the genomic nucleic acidsequence; insertion of one or more heterologous introns; deletion of oneor more upstream or downstream regulatory regions associated with thegenomic nucleic acid sequence; insertion of one or more heterologousupstream or downstream regulatory regions; deletion of the 5′ and/or 3′untranslated region associated with the genomic nucleic acid sequence;insertion of a heterologous 5′ and/or 3′ untranslated region; andmodification of a polyadenylation site. In some embodiments thenon-genomic nucleic acid molecule is a cDNA. In some embodiments thenon-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

Terms used to describe the protein structural motifs and secondarystructures herein have their standard meaning in the art. See forexample: Creighton, Thomas A., Proteins: Structures and MolecularProperties, W.H. Freeman; Second ed. (1992); Kabsch, et. al., Dictionaryof Protein Secondary Structure, Biopolymers, Vol. 22, 2577-2637 (1983).

As used herein, the term “improved insecticidal activity” or “improvedpesticidal activity” refers to an insecticidal polypeptide of theembodiments that has enhanced insecticidal activity relative to theactivity of its corresponding wild-type protein, and/or an insecticidalpolypeptide that is effective against a broader range of insects, and/oran insecticidal polypeptide having specificity for an insect that is notsusceptible to the toxicity of the wild-type protein. A finding ofimproved or enhanced pesticidal activity requires a demonstration of anincrease of pesticidal activity of at least 10%, against the insecttarget, or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 100%,150%, 200%, or 300% or greater increase of pesticidal activity relativeto the pesticidal activity of the wild-type insecticidal polypeptidedetermined against the same insect.

For example, an improved pesticidal or insecticidal activity is providedwhere a wider or narrower range of insects controlled by the polypeptiderelative to the range of insects that is affected by a wild-type toxin.A wider range of control may be desirable where versatility is desired,while a narrower range may be desirable where, for example, beneficialinsects might otherwise be impacted by use or presence of the toxin.While the embodiments are not bound by any particular mechanism ofaction, an improved pesticidal activity may also be provided by changesin one or more characteristics of a polypeptide; for example, thestability or longevity of a polypeptide in an insect gut may beincreased relative to the stability or longevity of a correspondingwild-type protein.

Changes can be made to the polypeptides of the disclosure that conferother desirable physical or biological characteristics, including butnot limited to: reduced hemolytic activity, altered sensitivity topepsin, trypsin, chymotrypsin, and other proteases. Alterations that canbe made without a negative impact on the pesticidal or insecticidalactivity of the protein are encompassed by the disclosure.

For example, conservative amino acid substitutions may be made at one ormore predicted, nonessential, amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a polypeptide without altering the biological activity. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include: amino acids with basicside chains (e.g., lysine, arginine, histidine); acidic side chains(e.g., aspartic acid, glutamic acid); polar, negatively charged residuesand their amides (e.g., aspartic acid, asparagine, glutamic, acid,glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity,except as otherwise noted herein. Examples of residues that areconserved and that may be essential for protein activity include, forexample, residues that are identical between all proteins contained inan alignment of similar or related toxins to the sequences of theembodiments (e.g., residues that are identical in an alignment ofhomologous proteins). Examples of residues that are conserved but thatmay allow conservative amino acid substitutions and still retainactivity include, for example, residues that have only conservativesubstitutions between all proteins contained in an alignment of similaror related toxins to the sequences of the embodiments (e.g., residuesthat have only conservative substitutions between all proteins containedin the alignment homologous proteins). However, one of skill in the artwould understand that functional variants may have minor conserved ornonconserved alterations in the conserved residues. Guidance as toappropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model ofDayhoff, et al., (1978) Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, J Mol Biol. 157(1): 105-32,1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, Ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +0.2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein. As detailedin U.S. Pat. No. 4,554,101, the following hydrophilicity values havebeen assigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentpolypeptide coding regions can be used to create a new polypeptidepossessing the desired properties. In this manner, libraries ofrecombinant polynucleotides are generated from a population of relatedsequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredpolypeptides. Domains may be swapped between pesticidal polypeptides,resulting in hybrid or chimeric toxins with improved pesticidal activityor target spectrum. Methods for generating recombinant proteins andtesting them for pesticidal activity are well known in the art (see, forexample, Naimov, et al., (2001) Appl. Environ. Microbiol. 67:5328-5330;de Maagd, et al., (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge, etal., (1991) J. Biol. Chem. 266:17954-17958; Schnepf, et al., (1990) J.Biol. Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ.Microbiol. 65:2918-2925).

As used herein the term “reduced hemolytic activity” refers to aninsecticidal polypeptide of the embodiments that has decreased red bloodcell lysis activity relative to the activity of its correspondingwild-type protein. In some embodiments the insecticidal polypeptide ofthe embodiments has reduced hemolytic activity compared to the hemolyticactivity of the polypeptide of SEQ ID NO: 2. In some embodiments thehemolytic activity is decreased at least 1.5 fold, 2 fold, 3 fold, 4fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold,13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 foldor greater compared to the hemolytic activity of the polypeptide of SEQID NO: 2.

Amino acid sequences homologous to MP467 (SEQ ID NO: 2) were identifiedby similarity search on the non-redundant database (nr) of NationalCenter for Bioinformatics Information (NCBI) using BLAST and PSI-BLAST.Hidden Markov Model profile method (HMMER3) was also used to expand themembership search through two PFAM families, aerolysin and ETX_MT2(Clostridium ε-toxin and Bacillus mosquitocidal toxin). A total of 485sequences in the NCBI non-redundant database have a low, but detectable,similarity to the MP467 (<70%). After redundancy reduction in which twosequences are clustered as one if they are with 95% identical over 95%length, 333 unique sequences are identified. The homologous proteins arefound in all kingdoms of life although vast majority of them arebacterial toxins such as aerolysin from Aeromonas, alpha-toxin fromClostridium septicum, ε-toxin form Clostridium perfringens, parasporin-2(PS2) from Bacillus thuringiensis.

A hydralysin from Hydra viridissima showed 67% sequence similarity toMP467 when aligned over the entire length of MP467 (see FIG. 1, and SEQID NO: 4). This protein demonstrated weak insecticidal activity with asimilar spectrum.

A protein from Bacillus thuringiensis, parasporin-2Aa (Ito A. et al, J.Biol. Chem, 279:21282-21286 2004—Accession # BAC79010.1) showed ˜57%sequence similarity to MP467 (SEQ ID NO: 2) when aligned over the entirelength of MP467 (see FIG. 3 and SEQ ID NO: 10). Early publications namedsome of these proteins parasporins, to distinguish them from theinsecticidal Cry proteins. The parasporins are still thought to benon-insecticidal. Later, the parasporins adopted the Cry proteinnomenclature, hence multiple names for the same protein. As used herein,“parasporin-2Aa” “PS2” and “Cry46Aa” may be used interchangeably andrefer to SEQ ID NO: 10 and its functional variants and fragments.Contrary to the reports in the literature that indicated Cry46Aa (SEQ IDNO: 10) lacked insecticidal activity it was surprisingly demonstratedherein that Cry46Aa (SEQ ID NO: 10) had insecticidal activity over abroad range of insects (See Example 2). Additional Cry46A homologs haverecently been identified from Bacillus thuringiensis Strain A1470(Okumura S. et al, Biotechnol Lett 35:1889-1984, 2013—Accession #BAG68906.1) and Cry46Ab (SEQ ID NO: 12) from Bacillus thuringiensisstrain TK-E6 (Hayakawa T. et al, Current Microbiology 55:278-283,2007—Accession # BAD35170.1), which showed ˜53% sequence similarity(FIG. 2 and Table 7) to MP467 (SEQ ID NO: 2).

Two similar proteins from B. thuringiensis, MP543 (SEQ ID NO: 6) andMP544 (SEQ ID NO: 8) showing similar structural homology to MP467 (SEQID NO: 2) showed no insecticidal activity at the concentrations tested.An amino acid sequence alignment (FIG. 4) of MP543 (SEQ ID NO: 6), MP544(SEQ ID NO: 8), and MP467 (SEQ ID NO: 2), reveals that MP543 (SEQ ID NO:6) and MP544 (SEQ ID NO: 8) have amino acid substitutions in the type 1′β-turn compared to that of MP467 (SEQ ID NO: 2).

In addition, hydralysins from Cnidaria, hemolytic lectin from theparasitic mushroom Laetiporus suiphureus, and enterolobin produced bythe seeds of the Brazilian tree are also in this list.

One aspect of the disclosure pertains to isolated or recombinant nucleicacid molecules comprising nucleic acid sequences encoding thepolypeptides of the disclosure or biologically active portions thereof,as well as nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules encoding proteins with regionsof sequence homology. As used herein, the term “nucleic acid” isintended to include DNA molecules (e.g., recombinant DNA, cDNA, genomicDNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of a native(non-synthetic), endogenous sequence. A full-length polynucleotideencodes the full-length, catalytically active form of the specifiedprotein.

An “isolated” or “recombinant” nucleic acid molecule (or DNA) is usedherein to refer to a nucleic acid sequence (or DNA) that is no longer inits natural environment, for example in an in vitro or in a recombinantbacterial or plant host cell. In some embodiments, an “isolated” or“recombinant” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forpurposes of the disclosure, “isolated” or “recombinant” when used torefer to nucleic acid molecules excludes isolated chromosomes. Forexample, in various embodiments, the recombinant nucleic acid moleculeencoding a polypeptide of the disclosure can contain less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid sequencesthat naturally flank the nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived.

A variety of polynucleotides that encode a polypeptide of the disclosureor related proteins are contemplated. Such polynucleotides are usefulfor production of polypeptides in host cells when operably linked tosuitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encodepolypeptides of the disclosure or related proteins.

As used herein, the term “mutant nucleotide sequence” or “mutation” or“mutagenized nucleotide sequence” connotes a nucleotide sequence thathas been mutagenized or altered to contain one or more nucleotideresidues (e.g., base pair) that is not present in the correspondingwild-type sequence. Such mutagenesis or alteration consists of one ormore additions, deletions, or substitutions or replacements of nucleicacid residues. When mutations are made by adding, removing, or replacingan amino acid of a proteolytic site, such addition, removal, orreplacement may be within or adjacent to the proteolytic site motif, solong as the object of the mutation is accomplished (i.e., so long asproteolysis at the site is changed).

A mutant nucleotide sequence can encode a mutant insecticidal toxinshowing improved or decreased insecticidal activity, or an amino acidsequence which confers improved or decreased insecticidal activity on apolypeptide containing it. As used herein, the term “mutant” or“mutation” in the context of a protein a polypeptide or amino acidsequence refers to a sequence which has been mutagenized or altered tocontain one or more amino acid residues that are not present in thecorresponding wild-type sequence. Such mutagenesis or alterationconsists of one or more additions, deletions, or substitutions orreplacements of amino acid residues. A mutant polypeptide shows improvedor decreased insecticidal activity, or represents an amino acid sequencewhich confers improved insecticidal activity on a polypeptide containingit. Thus, the term “mutant” or “mutation” refers to either or both ofthe mutant nucleotide sequence and the encoded amino acids. Mutants maybe used alone or in any compatible combination with other mutants of theembodiments or with other mutants. A “mutant polypeptide” may converselyshow a decrease in insecticidal activity. Where more than one mutationis added to a particular nucleic acid or protein, the mutations may beadded at the same time or sequentially; if sequentially, mutations maybe added in any suitable order.

Thus, polypeptides encoded by nucleotide sequences comprising mutationswill comprise at least one amino acid change or addition relative to thenative or background sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,32, 35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280or more amino acid changes or additions. Pesticidal activity of apolypeptide may also be improved by truncation of the native orfull-length sequence, as is known in the art.

In this manner, embodiments of the disclosure provide amino acid andnucleic acid sequences comprising a variety of mutations, such as forexample, mutagenesis of the hydrophobic patch that retain or exceed thepesticidal activity of the wild type protein.

Compositions of the embodiments include nucleic acids, fragments, andvariants thereof that encode pesticidal polypeptides. In particular, theembodiments provide for isolated nucleic acid molecules comprisingnucleotide sequences encoding the amino acid sequence shown in SEQ IDNO: 2 and SEQ ID NO: 24, or the nucleotide sequences encoding said aminoacid sequence, for example the nucleotide sequence set forth in SEQ IDNO: 1, SEQ ID NO: 23 and SEQ ID NO: 28 insecticidal fragments andvariants thereof and insecticidal variants shown in Table 6.

Fragments and variants of the nucleotide and amino acid sequences andthe polypeptides encoded thereby are also encompassed by theembodiments. As used herein the term “fragment” refers to a portion of anucleotide sequence of a polynucleotide or a portion of an amino acidsequence of a polypeptide of the embodiments. Fragments of a nucleotidesequence may encode protein fragments that retain the biologicalactivity of the native or corresponding full-length protein and hencepossess pesticidal activity. Thus, it is acknowledged that some of thepolynucleotide and amino acid sequences of the embodiments can correctlybe referred to as both fragments and mutants.

It is to be understood that the term “fragment,” as it is used to referto nucleic acid sequences of the embodiments, also encompasses sequencesthat are useful as hybridization probes. This class of nucleotidesequences generally does not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence may rangefrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, and up to the full-length nucleotide sequence encoding theproteins of the embodiments.

A fragment of a nucleotide sequence of the embodiments that encodes abiologically active portion of a pesticidal protein of the embodimentswill encode at least 15, 25, 30, 50, 100, 200 or 300 contiguous aminoacids, or up to the total number of amino acids present in a pesticidalpolypeptide of the embodiments (for example, 258 amino acids for SEQ IDNO: 2). Thus, it is understood that the embodiments also encompasspolypeptides that are fragments of the exemplary pesticidal proteins ofthe embodiments and having lengths of at least 15, 25, 30, 50, 100, 200,or 300 contiguous amino acids, or up to the total number of amino acidspresent in a pesticidal polypeptide of the embodiments. Fragments of anucleotide sequence of the embodiments that are useful as hybridizationprobes or PCR primers generally need not encode a biologically activeportion of a pesticidal protein. Thus, a fragment of a nucleic acid ofthe embodiments may encode a biologically active portion of a pesticidalprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed herein. A biologicallyactive portion of a pesticidal protein can be prepared by isolating aportion of one of the nucleotide sequences of the embodiments,expressing the encoded portion of the pesticidal protein (e.g., byrecombinant expression in vitro), and assessing the activity of theencoded portion of the pesticidal protein.

Nucleic acids that are fragments of a nucleotide sequence of theembodiments comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 600, 700, or 800 nucleotides, or up to the number ofnucleotides present in a nucleotide sequence disclosed herein (forexample, 774 nucleotides for SEQ ID NO: 1). Particular embodimentsenvision fragments derived from (e.g., produced from) a first nucleicacid of the embodiments, wherein the fragment encodes a truncated toxincharacterized by pesticidal activity. Truncated polypeptides encoded bythe polynucleotide fragments of the embodiments are characterized bypesticidal activity that is either equivalent to, or improved, relativeto the activity of the corresponding full-length polypeptide encoded bythe first nucleic acid from which the fragment is derived. It isenvisioned that such nucleic acid fragments of the embodiments may betruncated at the 3′ end of the native or corresponding full-lengthcoding sequence. Nucleic acid fragments may also be truncated at boththe 5′ and 3′ end of the native or corresponding full-length codingsequence.

The term “variants” is used herein to refer to substantially similarsequences. For nucleotide sequences, conservative variants include thosesequences that, because of the degeneracy of the genetic code, encodethe amino acid sequence of one of the pesticidal polypeptides of theembodiments. Naturally occurring allelic variants such as these can beidentified with the use of well-known molecular biology techniques, suchas, for example, polymerase chain reaction (PCR) and hybridizationtechniques as outlined herein.

Variant nucleotide sequences also include synthetically derivednucleotide sequences, such as those generated, for example, by usingsite-directed mutagenesis but which still encode a pesticidal protein ofthe embodiments, such as a mutant toxin. Generally, variants of aparticular nucleotide sequence of the embodiments will have at leastabout 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to that particularnucleotide sequence as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A variant of anucleotide sequence of the embodiments may differ from that sequence byas few as 1-15 nucleotides, as few as 1-10, such as 6-10, as few as 5,as few as 4, 3, 2, or even 1 nucleotide.

Variants of a particular nucleotide sequence of the embodiments (i.e.,an exemplary nucleotide sequence) can also be evaluated by comparison ofthe percent sequence identity between the polypeptide encoded by avariant nucleotide sequence and the polypeptide encoded by the referencenucleotide sequence. Thus, for example, isolated nucleic acids thatencode a polypeptide with a given percent sequence identity to thepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 24 are disclosed. Percentsequence identity between any two polypeptides can be calculated usingsequence alignment programs described elsewhere herein using defaultparameters. Where any given pair of polynucleotides of the embodimentsis evaluated by comparison of the percent sequence identity shared bythe two polypeptides they encode, the percent sequence identity betweenthe two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%,65%, 70%, generally at least about 75%, 80%, 85%, at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least about 98%, 99% or moresequence identity.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from other organisms, particularly otherbacteria, and more particularly other Bacillus strains. In this manner,methods such as PCR, hybridization, and the like can be used to identifysuch sequences based on their sequence homology to the sequences setforth herein. Sequences that are selected based on their sequenceidentity to the entire sequences set forth herein or to fragmentsthereof are encompassed by the embodiments. Such sequences includesequences that are orthologs of the disclosed sequences. The term“orthologs” refers to genes derived from a common ancestral gene andwhich are found in different species as a result of speciation. Genesfound in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein. Functions of orthologsare often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.),hereinafter “Sambrook”. See also Innis et al., eds. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NewYork); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press,New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual(Academic Press, New York). Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequences of theembodiments. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook.

For example, an entire sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding sequences and messenger RNAs. To achievespecific hybridization under a variety of conditions, such probesinclude sequences that are unique to the sequences of the embodimentsand are generally at least about 10 or 20 nucleotides in length. Suchprobes may be used to amplify corresponding sequences from a chosenorganism by PCR. This technique may be used to isolate additional codingsequences from a desired organism or as a diagnostic assay to determinethe presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook).

Hybridization of such sequences may be carried out under stringentconditions. The term “stringent conditions” or “stringent hybridizationconditions” as used herein refers to conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold, 5-fold, or 10-fold overbackground). Stringent conditions are sequence-dependent and will bedifferent in different circumstances. By controlling the stringency ofthe hybridization and/or washing conditions, target sequences that are100% complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 or 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a final wash in 0.1×SSC at 60 to 65° C. for at least about20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1%SDS. The duration of hybridization is generally less than about 24hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) (thermal melting point)can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, “% form” isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. Washes are typicallyperformed at least until equilibrium is reached and a low backgroundlevel of hybridization is achieved, such as for 2 hours, 1 hour, or 30minutes.

T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with ≥90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the T_(m);moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the T_(m); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20 Clower than the T_(m).

Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), theSSC concentration can be increased so that a higher temperature can beused. An extensive guide to the hybridization of nucleic acids is foundin Tijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See also Sambrook. Thus, isolatedsequences that encode a protein of the embodiments and hybridize understringent conditions to the sequences disclosed herein, or to fragmentsthereof, are encompassed by the embodiments.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of theembodiments. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the embodiments. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See the National Center for Biotechnology Informationwebsite on the world wide web at ncbi.hlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. The term“equivalent program” as used herein refers to any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the GCG Wisconsin Genetics Software Package for protein sequences are8 and 2, respectively. For nucleotide sequences the default gap creationpenalty is 50 while the default gap extension penalty is 3. The gapcreation and gap extension penalties can be expressed as an integerselected from the group of integers consisting of from 0 to 200. Thus,for example, the gap creation and gap extension penalties can be 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%. 80%,90%, or 95% or more sequence identity when compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like. Substantial identity of amino acid sequences for thesepurposes generally means sequence identity of at least 60%, 70%, 80%,90%, or 95% or more sequence identity.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C. lower than the T_(m), depending upon thedesired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 80%,85%, 90%, 95%, or more sequence identity to a reference sequence over aspecified comparison window. Optimal alignment for these purposes can beconducted using the global alignment algorithm of Needleman and Wunsch(1970) supra. An indication that two peptide sequences are substantiallyidentical is that one peptide is immunologically reactive withantibodies raised against the second peptide. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. Peptides that are“substantially similar” share sequences as noted above except thatresidue positions that are not identical may differ by conservativeamino acid changes.

The use of the term “constructs” herein is not intended to limit theembodiments to nucleotide constructs comprising DNA. Those of ordinaryskill in the art will recognize that nucleotide constructs, particularlypolynucleotides and oligonucleotides composed of ribonucleotides andcombinations of ribonucleotides and deoxyribonucleotides, may also beemployed in the methods disclosed herein. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments additionallyencompass all complementary forms of such constructs, molecules, andsequences. Further, the nucleotide constructs, nucleotide molecules, andnucleotide sequences of the embodiments encompass all nucleotideconstructs, molecules, and sequences which can be employed in themethods of the embodiments for transforming plants including, but notlimited to, those comprised of deoxyribonucleotides, ribonucleotides,and combinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs, nucleic acids, and nucleotide sequences of theembodiments also encompass all forms of nucleotide constructs including,but not limited to, single-stranded forms, double-stranded forms,hairpins, stem-and-loop structures, and the like.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, “operablylinked” means that the nucleic acid sequences being linked arecontiguous and, where necessary to join two protein coding regions,contiguous and in the same reading frame. The construct may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple DNA constructs.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the insecticidal sequence to be under thetranscriptional regulation of the regulatory regions. The DNA constructmay additionally contain selectable marker genes.

The DNA construct may include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host, or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray et al., supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli, oreukaryotic cells such as yeast, insect, amphibian, or mammalian cells,or monocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes or constructs may additionally contain 5′leader sequences. Such leader sequences can act to enhance translation.Translation leaders are known in the art and include: picornavirusleaders, for example, EMCV leader (Encephalomyocarditis 5′ noncodingregion) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco EtchVirus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (MaizeDwarf Mosaic Virus), human immunoglobulin heavy-chain binding protein(BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leaderfrom the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Joblinget al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV)(Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, NewYork), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81: 382-385). See also, Della-Cioppa etal. (1987) Plant Physiol. 84: 965-968.

In preparing the expression cassette or construct, the various DNAfragments may be manipulated so as to provide for the DNA sequences inthe proper orientation and, as appropriate, in the proper reading frame.Toward this end, adapters or linkers may be employed to join the DNAfragments or other manipulations may be involved to provide forconvenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible, orother promoters for expression in the host organism. Suitableconstitutive promoters for use in a plant host cell include, forexample, the core promoter of the Rsyn7 promoter and other constitutivepromoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the coreCaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin(McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen etal. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992)Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor. Appl.Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Other constitutivepromoters include, for example, those discussed in U.S. Pat. Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680;5,268,463; 5,608,142; and 6,177,611.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev. Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology 14: 494-498); wun1 andwun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford et al. (1989)Mol. Gen. Genet. 215: 200-208); systemin (McGurl et al. (1992) Science225: 1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene(Corderok et al. (1994) Plant J. 6(2): 141-150); and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89: 245-254; Uknes et al. (1992) Plant Cell 4: 645-656; and VanLoon (1985) Plant Mol. Virol. 4: 111-116. See also WO 99/43819, hereinincorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced pesticidalprotein expression within a particular plant tissue. Tissue-preferredpromoters include those discussed in Yamamoto et al. (1997) Plant J.12(2) 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire et al. (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also Bogusz et al.(1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a β-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi (1991) describe their analysis of the promoters of the highlyexpressed roIC and roID root-inducing genes of Agrobacterium rhizogenes(see Plant Science (Limerick) 79(1):69-76). They concluded that enhancerand tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri et al. (1989) used gene fusion to lacZ to show that theAgrobacterium T-DNA gene encoding octopine synthase is especially activein the epidermis of the root tip and that the TR2′ gene is root specificin the intact plant and stimulated by wounding in leaf tissue, anespecially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The TR1′gene fused to nptII (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and mi1ps(myo-inositol-1-phosphate synthase) (see U.S. Pat. No. 6,225,529, hereinincorporated by reference). Gamma-zein and Glb-1 are endosperm-specificpromoters. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference.

A promoter that has “preferred” expression in a particular tissue isexpressed in that tissue to a greater degree than in at least one otherplant tissue. Some tissue-preferred promoters show expression almostexclusively in the particular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142; and 6,177,611; herein incorporated by reference.

Generally, the expression cassette or construct will comprise aselectable marker gene for the selection of transformed cells.Selectable marker genes are utilized for the selection of transformedcells or tissues. Marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO)and hygromycin phosphotransferase (HPT), as well as genes conferringresistance to herbicidal compounds, such as glufosinate ammonium,bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).Additional examples of suitable selectable marker genes include, but arenot limited to, genes encoding resistance to chloramphenicol (HerreraEstrella et al. (1983) EMBO J. 2:987-992); methotrexate (HerreraEstrella et al. (1983) Nature 303:209-213; and Meijer et al. (1991)Plant Mol. Biol. 16:807-820); streptomycin (Jones et al. (1987) Mol.Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996)Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol.Biol. 7:171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker et al. (1988) Science 242:419-423);glyphosate (Shaw et al. (1986) Science 233:478-481; and U.S. applicationSer. Nos. 10/004,357; and 10/427,692); phosphinothricin (DeBlock et al.(1987) EMBO J. 6:2513-2518). See generally, Yarranton (1992) Curr. Opin.Biotech. 3: 506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci.USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992)Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines etal. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990)Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl.Acad. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci.USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin(1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992)Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); andGill et al. (1988) Nature 334: 721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

A further embodiment relates to a transformed organism such as anorganism selected from the group consisting of plant and insect cells,bacteria, yeast, baculoviruses, protozoa, nematodes, and algae. Thetransformed organism comprises: a DNA molecule of the embodiments, anexpression cassette comprising the said DNA molecule, or a vectorcomprising the said expression cassette, which may be stablyincorporated into the genome of the transformed organism.

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4: 320-334), electroporation(Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782; Tomes et al.(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al.(1988) Biotechnology 6: 923-926); and Lecl transformation (WO 00/28058).For potato transformation see Tu et al. (1998) Plant Molecular Biology37: 829-838 and Chong et al. (2000) Transgenic Research 9: 71-78.Additional transformation procedures can be found in Weissinger et al.(1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) ParticulateScience and Technology 5: 27-37 (onion); Christou et al. (1988) PlantPhysiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice);Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (maize);Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos.5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol.91: 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc.Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) inThe Experimental Manipulation of Ovule Tissues, ed. Chapman et al.(Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84: 560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the Cry toxin protein or variants and fragmentsthereof directly into the plant or the introduction of the Cry toxintranscript into the plant. Such methods include, for example,microinjection or particle bombardment. See, for example, Crossway etal. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci.44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 andHush et al. (1994) The Journal of Cell Science 107: 775-784, all ofwhich are herein incorporated by reference. Alternatively, the Cry toxinpolynucleotide can be transiently transformed into the plant usingtechniques known in the art. Such techniques include viral vector systemand the precipitation of the polynucleotide in a manner that precludessubsequent release of the DNA. Thus, transcription from theparticle-bound DNA can occur, but the frequency with which it isreleased to become integrated into the genome is greatly reduced. Suchmethods include the use of particles coated with polyethylimine (PEI;Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the embodiments can be contained in transfercassette flanked by two non-identical recombination sites. The transfercassette is introduced into a plant have stably incorporated into itsgenome a target site which is flanked by two non-identical recombinationsites that correspond to the sites of the transfer cassette. Anappropriate recombinase is provided and the transfer cassette isintegrated at the target site. The polynucleotide of interest is therebyintegrated at a specific chromosomal position in the plant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5: 81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired pesticidal protein. It isalso recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of a pesticidal protein of theembodiments, may have the desired pesticidal activity. Such viralpolyproteins and the nucleotide sequences that encode for them areencompassed by the embodiments. Methods for providing plants withnucleotide constructs and producing the encoded proteins in the plants,which involve viral DNA or RNA molecules are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367; and5,316,931; herein incorporated by reference.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves, and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannus); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewings fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithii); Bermuda grass (Cynodon spp.); St. Augustine grass(Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass(Paspalum notatum); carpet grass (Axonopus affinis); centipede grass(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);seashore paspalum (Paspalum vaginatum); blue gramma (Boutelouagracilis); buffalo grass (Buchloe dactyloids); sideoats gramma(Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

In certain embodiments the nucleic acid sequences of the embodiments canbe stacked with any combination of polynucleotide sequences of interestin order to create plants with a desired phenotype. For example, thepolynucleotides of the embodiments may be stacked with any otherpolynucleotides encoding polypeptides having pesticidal and/orinsecticidal activity, such as Bt toxic proteins (described in U.S. Pat.Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiseret al. (1986) Gene 48:109), pentin (described in U.S. Pat. No.5,981,722) and the like. The combinations generated can also includemultiple copies of any one of the polynucleotides of interest. Thepolynucleotides of the embodiments can also be stacked with any othergene or combination of genes to produce plants with a variety of desiredtrait combinations including but not limited to traits desirable foranimal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802; and 5,703,049); barley high lysine (Williamson etal. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122) and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279;Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) PlantMol. Biol. 12: 123)); increased digestibility (e.g., modified storageproteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); andthioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001)), the disclosures of which are herein incorporated by reference.

The polynucleotides of the embodiments can also be stacked with traitsdesirable for disease or herbicide resistance (e.g., fumonisindetoxification genes (U.S. Pat. No. 5,792,931); avirulence and diseaseresistance genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene and GAT gene as disclosed in U.S. applicationSer. Nos. 10/004,357; and 10/427,692); and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170: 5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe embodiments with polynucleotides providing agronomic traits such asmale sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength,flowering time, or transformation technology traits such as cell cycleregulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO99/25821), the disclosures of which are herein incorporated byreference.

These stacked combinations can be created by any method including butnot limited to cross breeding plants by any conventional or TOPCROSS®methodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference.

Compositions of the embodiments find use in protecting plants, seeds,and plant products in a variety of ways. For example, the compositionscan be used in a method that involves placing an effective amount of thepesticidal composition in the environment of the pest by a procedureselected from the group consisting of spraying, dusting, broadcasting,or seed coating.

Before plant propagation material (fruit, tuber, bulb, corm, grains,seed), but especially seed, is sold as a commercial product, it iscustomarily treated with a protectant coating comprising herbicides,insecticides, fungicides, bactericides, nematocides, molluscicides, ormixtures of several of these preparations, if desired together withfurther carriers, surfactants, or application-promoting adjuvantscustomarily employed in the art of formulation to provide protectionagainst damage caused by bacterial, fungal, or animal pests. In order totreat the seed, the protectant coating may be applied to the seedseither by impregnating the tubers or grains with a liquid formulation orby coating them with a combined wet or dry formulation. In addition, inspecial cases, other methods of application to plants are possible,e.g., treatment directed at the buds or the fruit.

The plant seed of the embodiments comprising a nucleotide sequenceencoding a pesticidal protein of the embodiments may be treated with aseed protectant coating comprising a seed treatment compound, such as,for example, captan, carboxin, thiram, methalaxyl, pirimiphos-methyl,and others that are commonly used in seed treatment. In one embodiment,a seed protectant coating comprising a pesticidal composition of theembodiments is used alone or in combination with one of the seedprotectant coatings customarily used in seed treatment.

It is recognized that the genes encoding the pesticidal proteins can beused to transform insect pathogenic organisms. Such organisms includebaculoviruses, fungi, protozoa, bacteria, and nematodes.

A gene encoding a pesticidal protein of the embodiments may beintroduced via a suitable vector into a microbial host, and said hostapplied to the environment, or to plants or animals. The term“introduced” in the context of inserting a nucleic acid into a cell,means “transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrialDNA), converted into an autonomous replicon, or transiently expressed(e.g., transfected mRNA).

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the pesticidalprotein, and desirably, provide for improved protection of the pesticidefrom environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas campestris,Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli andAzotobacter vinlandir and phytosphere yeast species such as Rhodotorularubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Of particular interest are the pigmentedmicroorganisms.

A number of ways are available for introducing a gene expressing thepesticidal protein into the microorganism host under conditions thatallow for stable maintenance and expression of the gene. For example,expression cassettes can be constructed which include the nucleotideconstructs of interest operably linked with the transcriptional andtranslational regulatory signals for expression of the nucleotideconstructs, and a nucleotide sequence homologous with a sequence in thehost organism, whereby integration will occur, and/or a replicationsystem that is functional in the host, whereby integration or stablemaintenance will occur.

Transcriptional and translational regulatory signals include, but arenot limited to, promoters, transcriptional initiation start sites,operators, activators, enhancers, other regulatory elements, ribosomalbinding sites, an initiation codon, termination signals, and the like.See, for example, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2;Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, ed.Maniatis et al. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.), hereinafter “Sambrook II”; Davis et al., eds. (1980)Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press), ColdSpring Harbor, N.Y.; and the references cited therein.

Suitable host cells, where the pesticidal protein-containing cells willbe treated to prolong the activity of the pesticidal proteins in thecell when the treated cell is applied to the environment of the targetpest(s), may include either prokaryotes or eukaryotes, normally beinglimited to those cells that do not produce substances toxic to higherorganisms, such as mammals. However, organisms that produce substancestoxic to higher organisms could be used, where the toxin is unstable orthe level of application sufficiently low as to avoid any possibility oftoxicity to a mammalian host. As hosts, of particular interest will bethe prokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter Azotobacteraceae and Nitrobacteraceae. Amongeukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of pesticidal protein production include ease of introducingthe pesticidal protein gene into the host, availability of expressionsystems, efficiency of expression, stability of the protein in the host,and the presence of auxiliary genetic capabilities. Characteristics ofinterest for use as a pesticide microcapsule include protectivequalities for the pesticide, such as thick cell walls, pigmentation, andintracellular packaging or formation of inclusion bodies; leaf affinity;lack of mammalian toxicity; attractiveness to pests for ingestion; easeof killing and fixing without damage to the toxin; and the like. Otherconsiderations include ease of formulation and handling, economics,storage stability, and the like.

Host organisms of particular interest include yeast, such as Rhodotorulaspp., Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens), Erwinia spp., andFlavobacterium spp., and other such organisms, including Bt, E. coli,Bacillus subtilis, and the like.

Genes encoding the pesticidal proteins of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver pesticidal proteins to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the pesticidal proteins of the embodimentscan be introduced into a root-colonizing Bacillus cereus by standardmethods known in the art.

Genes encoding pesticidal proteins can be introduced, for example, intothe root-colonizing Bacillus by means of electro transformation.Specifically, genes encoding the pesticidal proteins can be cloned intoa shuttle vector, for example, pHT3101 (Lerecius et al. (1989) FEMSMicrobiol. Letts. 60: 211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular pesticidal protein gene can, forexample, be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius et al. (1989) FEMS Microbiol. Letts. 60:211-218).

Expression systems can be designed so that pesticidal proteins aresecreted outside the cytoplasm of gram-negative bacteria, such as E.coli, for example. Advantages of having pesticidal proteins secretedare: (1) avoidance of potential cytotoxic effects of the pesticidalprotein expressed; and (2) improvement in the efficiency of purificationof the pesticidal protein, including, but not limited to, increasedefficiency in the recovery and purification of the protein per volumecell broth and decreased time and/or costs of recovery and purificationper unit protein.

Pesticidal proteins can be made to be secreted in E. coli, for example,by fusing an appropriate E. coli signal peptide to the amino-terminalend of the pesticidal protein. Signal peptides recognized by E. coli canbe found in proteins already known to be secreted in E. coli, forexample the OmpA protein (Ghrayeb et al. (1984) EMBO J, 3:2437-2442).OmpA is a major protein of the E. coli outer membrane, and thus itssignal peptide is thought to be efficient in the translocation process.Also, the OmpA signal peptide does not need to be modified beforeprocessing as may be the case for other signal peptides, for examplelipoprotein signal peptide (Duffaud et al. (1987) Meth. Enzymol. 153:492).

Pesticidal proteins of the embodiments can be fermented in a bacterialhost and the resulting bacteria processed and used as a microbial sprayin the same manner that Bt strains have been used as insecticidalsprays. In the case of a pesticidal protein(s) that is secreted fromBacillus, the secretion signal is removed or mutated using proceduresknown in the art. Such mutations and/or deletions prevent secretion ofthe pesticidal protein(s) into the growth medium during the fermentationprocess. The pesticidal proteins are retained within the cell, and thecells are then processed to yield the encapsulated pesticidal proteins.Any suitable microorganism can be used for this purpose. Pseudomonas hasbeen used to express Bt toxins as encapsulated proteins and theresulting cells processed and sprayed as an insecticide (Gaertner et al.(1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the pesticidal proteins are produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated pesticidal proteins may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

In the embodiments, a transformed microorganism (which includes wholeorganisms, cells, spore(s), pesticidal protein(s), pesticidalcomponent(s), pest-impacting component(s), mutant(s), living or deadcells and cell components, including mixtures of living and dead cellsand cell components, and including broken cells and cell components) oran isolated pesticidal protein can be formulated with an acceptablecarrier into a pesticidal composition(s) that is, for example, asuspension, a solution, an emulsion, a dusting powder, a dispersiblegranule or pellet, a wettable powder, and an emulsifiable concentrate,an aerosol or spray, an impregnated granule, an adjuvant, a coatablepaste, a colloid, and also encapsulations in, for example, polymersubstances. Such formulated compositions may be prepared by suchconventional means as desiccation, lyophilization, homogenization,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematocides,molluscicides, acaricides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the embodiments are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the embodiments may be appliedto grain in preparation for or during storage in a grain bin or silo,etc. The compositions of the embodiments may be applied simultaneouslyor in succession with other compounds. Methods of applying an activeingredient of the embodiments or an agrochemical composition of theembodiments that contains at least one of the pesticidal proteinsproduced by the bacterial strains of the embodiments include, but arenot limited to, foliar application, seed coating, and soil application.The number of applications and the rate of application depend on theintensity of infestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; a carboxylateof a long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate of dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the embodiments can be in a suitable form for directapplication or as a concentrate of primary composition that requiresdilution with a suitable quantity of water or other diluent beforeapplication. The pesticidal concentration will vary depending upon thenature of the particular formulation, specifically, whether it is aconcentrate or to be used directly. The composition contains 1 to 98% ofa solid or liquid inert carrier, and 0 to 50% or 0.1 to 50% of asurfactant. These compositions will be administered at the labeled ratefor the commercial product, for example, about 0.01 lb-5.0 lb. per acrewhen in dry form and at about 0.01 pts.-10 pts. per acre when in liquidform.

In a further embodiment, the compositions, as well as the transformedmicroorganisms and pesticidal proteins of the embodiments, can betreated prior to formulation to prolong the pesticidal activity whenapplied to the environment of a target pest as long as the pretreatmentis not deleterious to the pesticidal activity. Such treatment can be bychemical and/or physical means as long as the treatment does notdeleteriously affect the properties of the composition(s). Examples ofchemical reagents include but are not limited to halogenating agents;aldehydes such as formaldehyde and glutaraldehyde; anti-infectives, suchas zephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.).

The compositions (including the transformed microorganisms andpesticidal proteins of the embodiments) can be applied to theenvironment of an insect pest by, for example, spraying, atomizing,dusting, scattering, coating or pouring, introducing into or on thesoil, introducing into irrigation water, by seed treatment or generalapplication or dusting at the time when the pest has begun to appear orbefore the appearance of pests as a protective measure. For example, thepesticidal protein and/or transformed microorganisms of the embodimentsmay be mixed with grain to protect the grain during storage. It isgenerally important to obtain good control of pests in the early stagesof plant growth, as this is the time when the plant can be most severelydamaged. The compositions of the embodiments can conveniently containanother insecticide if this is thought necessary. In one embodiment, thecomposition is applied directly to the soil, at a time of planting, ingranular form of a composition of a carrier and dead cells of a Bacillusstrain or transformed microorganism of the embodiments. Anotherembodiment is a granular form of a composition comprising anagrochemical such as, for example, an herbicide, an insecticide, afertilizer, an inert carrier, and dead cells of a Bacillus strain ortransformed microorganism of the embodiments.

In some embodiments the composition is a “non-naturally occurring”composition. As used herein a “non-naturally occurring” compositionrefers to a composition that is not found in nature. Such non-naturallyoccurring compositions include but are not limited to a composition thatcomprises a polynucleotide of the disclosure or a polypeptide of thedisclosure and at least one component not normally associated in naturewith a polynucleotide of the disclosure or a polypeptide of thedisclosure. Such non-naturally occurring compositions include but arenot limited to a plant or microorganism, excluding the plant ormicroorganism from which the polynucleotide of the disclosure or thepolypeptide of the disclosure was isolated from or derived from,transformed with a polynucleotide of the disclosure or comprising apolypeptide of the disclosure.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery, ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera.

Larvae and adults of the order Coleoptera include weevils from thefamilies Anthribidae, Bruchidae, and Curculionidae (including, but notlimited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrusoryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus(granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctataFabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte(sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seedweevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorusmaidis Chittenden (maize billbug)); flea beetles, cucumber beetles,rootworms, leaf beetles, potato beetles, and leafminers in the familyChrysomelidae (including, but not limited to: Leptinotarsa decemlineataSay (Colorado potato beetle); Diabrotica virgifera virgifera LeConte(western corn rootworm); D. barberi Smith & Lawrence (northern cornrootworm); D. undecimpunctata howardi Barber (southern corn rootworm);Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotretacruciferae Goeze (corn flea beetle); Colaspis brunnea Fabricius (grapecolaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogrammaexclamationis Fabricius (sunflower beetle)); beetles from the familyCoccinellidae (including, but not limited to: Epilachna varivestisMulsant (Mexican bean beetle)); chafers and other beetles from thefamily Scarabaeidae (including, but not limited to: Popillia japonicaNewman (Japanese beetle); Cyclocephala borealis Arrow (northern maskedchafer, white grub); C. immaculata Olivier (southern masked chafer,white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers, and heliothines in the family NoctuidaeSpodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms, and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermüller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakSilk moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Collas eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonotycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J.E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermüller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Adults and immatures of the order Diptera include: leafminers such asAgromyza parvicomis Loew (corn blotch leafminer); midges (including, butnot limited to: Contarinia sorghicola Coquillett (sorghum midge);Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Géhin(wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seedmidge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (fritflies); maggots (including, but not limited to: Delia platura Meigen(seedcorn maggot); D. coarctata Fallen (wheat bulb fly); and other Deliaspp., Meromyza americana Fitch (wheat stem maggot); Musca domesticaLinnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis Stein(lesser house flies); Stomoxys calcitrans Linnaeus (stable flies)); faceflies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; and othermuscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilusspp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysopsspp.; Melophagus ovinus Linnaeus (keds); and other Brachycera,mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black fliesProsimulium spp.; Simulium spp.; biting midges, sand flies, sciarids,and other Nematocera.

Adults and nymphs of the orders Hemiptera and Homoptera include insectssuch as, but not limited to, adelgids from the family Adelgidae, plantbugs from the family Miridae, cicadas from the family Cicadidae,leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppersfrom the families Cixiidae, Flatidae, Fulgoroidea, lssidae andDelphacidae, treehoppers from the family Membracidae, psyllids from thefamily Psyllidae, whiteflies from the family Aleyrodidae, aphids fromthe family Aphididae, phylloxera from the family Phylloxeridae,mealybugs from the family Pseudococcidae, scales from the familiesAsterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae,Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from thefamily Tingidae, stink bugs from the family Pentatomidae, cinch bugs,Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugsfrom the family Cercopidae squash bugs from the family Coreidae, and redbugs and cotton stainers from the family Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid); and T. citricida Kirkaldy (browncitrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande(pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly,sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleafwhitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodesabutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood(greenhouse whitefly); Empoasca fabae Harris (potato leafhopper);Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestesquadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler(green leafhopper); N. nigropictus Stål (rice leafhopper); Nilaparvatalugens Stål (brown planthopper); Peregrinus maidis Ashmead (cornplanthopper); Sogatella furcifera Horvath (white-backed planthopper);Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee(white apple leafhopper); Erythroneoura spp. (grape leafhoppers);Magicicada septendecim Linnaeus (periodical cicada); Icerya purchasiMaskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock(San Jose scale); Planococcus citri Risso (citrus mealybug);Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster(pear psylla); Trioza diospyri Ashmead (persimmon psylla).

Agronomically important species from the order Hemiptera include, butare not limited to: Acrosternum hilare Say (green stink bug); Anasatristis De Geer (squash bug); Blissus leucopterus leucopterus Say(chinch bug); Corythuca gossypii Fabricius (cotton lace bug);Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schïffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Insects included in the order Hemiptera include: Calocoris norvegicusGmelin (strawberry bug); Orthops campestris Linnaeus; Plesiocorisrugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomatobug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatusReuter (whitemarked fleahopper); Diaphnocoris chlorionis Say(honeylocust plant bug); Labopidicola allii Knight (onion plant bug);Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocorisrapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius(four-lined plant bug); Nysius ericae Schilling (false chinch bug);Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus(Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.;and Cimicidae spp.

Adults and larvae of the order Acari (mites) include: Aceria tosichellaKeifer (wheat curl mite); Petrobia latens Müller (brown wheat mite);spider mites and red mites in the family Tetranychidae, Panonychus ulmiKoch (European red mite); Tetranychus urticae Koch (two spotted spidermite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval(carmine spider mite); T. turkestani Ugarov & Nikolski (strawberryspider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisiMcGregor (citrus flat mite); rust and bud mites in the familyEriophyidae and other foliar feeding mites and mites important in humanand animal health, i.e. dust mites in the family Epidermoptidae,follicle mites in the family Demodicidae, grain mites in the familyGlycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deertick); I. holocyclus Neumann (Australian paralysis tick); Dermacentorvariabilis Say (American dog tick); Amblyomma americanum Linnaeus (lonestar tick); and scab and itch mites in the families Psoroptidae,Pyemotidae, and Sarcoptidae.

Insect pests of the order Thysanura include Lepisma saccharina Linnaeus(silverfish); Thermobia domestica Packard (firebrat). Additionalarthropod pests include: spiders in the order Araneae such as Loxoscelesreclusa Gertsch & Mulaik (brown recluse spider); and the Latrodectusmactans Fabricius (black widow spider); and centipedes in the orderScutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

TABLE 1 Insect pests and their abbreviations as used herein: ECBEuropean corn borer (Ostrinia nubilalis) FAW Fall armyworm (Spodopterafrugiperda) CEW Corn earworm (Helicoverpa zea Boddie) BCW Black cutworm(Agrotis ipsilon Hufnagel) SBL Soybean looper (Pseudoplusia includensWalker) VBC Velvetbean Caterpillar (Anticarsia gemmatalis Hübner) WCRWWestern Corn Rootworm (Diabrotica virgifera virgifera) StinkbugHalyomorpha halys, Acrostemum hilare, other Pentatomidae agriculturalpests Lygus Lygus Hesperus, L. elisus

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C. D. S. Tomlin Ed., Published by the British CropProduction Council, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

In some embodiments methods are provided for killing or controlling aninsect pest, comprising contacting the insect pest, eithersimultaneously or sequentially, with an insecticidally-effective amountof a recombinant polypeptide of the disclosure. In some embodimentsmethods are provided for killing an insect pest, comprising contactingthe insect pest with an insecticidally-effective amount of a recombinantpesticidal protein of SEQ ID NO: 2, SEQ ID NO: 24 or SEQ ID NO: 10 or avariant thereof, including but not limited to the polypeptides of SEQ IDNO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, and SEQ ID NO: 69.

As used herein, “controlling a pest population” or “controls a pest”refers to any effect on a pest that results in limiting the damage thatthe pest causes. Controlling a pest includes, but is not limited to:killing the pest, inhibiting development of the pest, altering fertilityor growth of the pest in such a manner that the pest provides lessdamage to the plant, decreasing the number of offspring produced,producing less fit pests, producing pests more susceptible to predatorattack or deterring the pests from eating the plant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population, either simultaneously or sequentially, with aninsecticidally-effective amount of a recombinant polypeptide of thedisclosure. In some embodiments methods are provided for controlling aninsect pest population resistant to a pesticidal protein, comprisingcontacting the insect pest population with an insecticidally-effectiveamount of a recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID NO:24 or SEQ ID NO: 10 or a variant thereof, including but not limited tothe polypeptides of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ IDNO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ ID NO: 69.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding a polypeptide of the disclosure. Insome embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding pesticidal protein of SEQ ID NO: 2,SEQ ID NO: 24 or SEQ ID NO: 10 or variants thereof, including but notlimited to the polypeptides of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ IDNO: 69.

In some embodiments, a pest population may be controlled by means of acomposition comprising the polypeptides of the disclosure in apesticidally-effective amount in a form including, but not limited to: apowder, dust, pellet, granule, spray, emulsion, colloid, or solution anda suitable carrier.

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, insectshave evolved that are resistant to B. thuringiensis δ-endotoxinsexpressed in transgenic plants. Such resistance, should it becomewidespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis δ-endotoxins.

One way to increasing the effectiveness of transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006. htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests would be to have a repository ofinsecticidal genes that are effective against groups of insect pests andwhich manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the polypeptides of the disclosure are useful as aninsect resistance management strategy in combination (i.e., pyramided)with other pesticidal proteins include but are not limited to Bt toxins,Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, and the like.

Provided are methods of controlling insect infestation(s) in atransgenic plant that promote insect resistance management, comprisingexpressing in the plant at least two different insecticidal proteinshaving different modes of action.

In some embodiments the methods of controlling insect infestation in atransgenic plant and promoting insect resistance management, at leastone of the insecticidal proteins comprise a polypeptide of thedisclosure insecticidal to insects.

In some embodiments the methods of controlling insect infestation in atransgenic plant and promoting insect resistance management, at leastone of the insecticidal proteins comprises a protein of SEQ ID NO: 2,SEQ ID NO: 24 or SEQ ID NO: 10 or variants thereof, including but notlimited to the polypeptides of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45,SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ IDNO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and SEQ IDNO: 69, insecticidal to insects in the order Hemiptera, Lepidopteraand/or Coleoptera.

In some embodiments the methods of controlling insect infestation in atransgenic plant and promoting insect resistance management, compriseexpressing in the transgenic plant a polypeptide of the disclosure andan insecticidal Cry protein having different modes of action.

Also provided are methods of reducing the likelihood of emergence ofinsect resistance to transgenic plants by expressing in the plantsinsecticidal proteins to control the insect species, comprisingexpression of a polypeptide of the disclosure in combination with asecond insecticidal protein to the insect species having different modesof action.

Also provided are means for effective insect resistance management oftransgenic plants, comprising co-expressing at high levels in theplants, two or more insecticidal proteins toxic to Hemiptera,Lepidoptera and/or Coleoptera insects but each exhibiting a differentmode of effectuating its killing activity, wherein the two or moreinsecticidal proteins comprise a polypeptide of the disclosure and a Cryprotein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing insecticidalproteins, comprising the step of referring to, submitting or relying oninsect assay binding data showing that the polypeptides of thedisclosure do not compete with binding sites for Cry proteins in suchinsects.

TABLE 2 Table of Sequences SEQ Sequence ID NO Gene Name Organism Type 1MP467 Bacillus sphaericus NT 2 ″ ″ AA 3 hydralysin-2 Hydra vulgaris NT 4″ ″ AA 5 MP543 B. Thuringiensis NT 6 ″ ″ AA 7 MP544 B. thuringiensis NT8 ″ ″ AA 9 Cry46Aa/(parasporin-2Aa) B. thuringiensis NT 10 ″ ″ AA 11Cry46Ab/(parasporin 2Ab) B. thuringiensis NT 12 ″ ″ AA 13 1′ beta turnmotif Artificial sequence AA 14 MP467 beta hairpin region Artificialsequence AA 15 Parasporin-2 beta hairpin Artificial sequence AA region16 Hydralysin beta hairpin Artificial sequence AA region 17 A-toxin betahairpin region Artificial sequence AA 18 aerolysin beta hairpin regionArtificial sequence AA 19 ε-toxin beta hairpin region Artificialsequence AA 20 hemolytic beta hairpin region Artificial sequence AA 21Enterotoxin beta hairpin Artificial sequence AA region 22 α-hemolysinbeta hairpin Artificial sequence AA region 23 MP812 B. Thuringiensis NA24 ″ ″ AA 25 Hemolytic patch motif Artificial sequence AA 26 Hemolyticpatch motif Artificial sequence AA 27 Hemolytic patch motif Artificialsequence AA 28 MP467 MODA Artificial sequence AA 29 MP467 W208AArtificial sequence AA 30 MP467 D204A Artificial sequence AA 31 MP467H205A Artificial sequence AA 32 MP467 Y206A Artificial sequence AA 33MP467 F207A Artificial sequence AA 34 MP467 F209A Artificial sequence AA35 MP467 Y56A Artificial sequence AA 36 MP467 H58A Artificial sequenceAA 37 MP467 W208R Artificial sequence AA 38 MP467 W208N Artificialsequence AA 39 MP467 W208D Artificial sequence AA 40 MP467 W208CArtificial sequence AA 41 MP467 W208Q Artificial sequence AA 42 MP467W208E Artificial sequence AA 43 MP467 W208G Artificial sequence AA 44MP467 W208H Artificial sequence AA 45 MP467 W208I Artificial sequence AA46 MP467 W208L Artificial sequence AA 47 MP467 W208K Artificial sequenceAA 48 MP467 W208M Artificial sequence AA 49 MP467 W208F Artificialsequence AA 50 MP467 W208S Artificial sequence AA 51 MP467 W208TArtificial sequence AA 52 MP467 W208Y Artificial sequence AA 53 MP467W208V Artificial sequence AA 54 MP467 Y206N-W208M Artificial sequence AA55 MP467 Y206F-W208M Artificial sequence AA 56 MP467 Y206W-W208MArtificial sequence AA 57 MP467 Y206R Artificial sequence AA 58 MP467Y206N Artificial sequence AA 59 MP467 Y206D Artificial sequence AA 60MP467 Y206Q Artificial sequence AA 61 MP467 Y206H Artificial sequence AA62 MP467 Y206I Artificial sequence AA 63 MP467 Y206L Artificial sequenceAA 64 MP467 Y206M Artificial sequence AA 65 MP467 Y206F Artificialsequence AA 66 MP467 Y206S Artificial sequence AA 67 MP467 Y206TArtificial sequence AA 68 MP467 Y206W Artificial sequence AA 69 MP467Y206V Artificial sequence AA

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTALS Example 1: Isolation and Modeling of MP467 and Cry46Aa

The insecticidal protein MP467 (SEQ ID NO: 2) was obtained from a screenof proteins derived from the Bacillus sphaericus strain AM1922.

MP467 ortholog sequences were identified by similarity search on thenon-redundant database (nr) of National Center for BioinformaticsInformation (NCBI) using BLAST and PSI-BLAST. Hidden Markov Modelprofile method (HMMER3) was also used to expand the membership searchthrough two PFAM families, aerolysin and ETX_MT2 (Clostridium ε-toxinand Bacillus mosquitocidal toxin). A total 485 sequences in the NCBInon-redundant database have a detectable similarity to the MP467. Afterredundancy reduction in which two sequences are clustered as one if theyare with 95% identical over 95% length, 333 unique sequences areidentified. The ortholog proteins were found in all kingdoms of lifealthough vast majority of them are bacterial toxins such as aerolysinfrom Aeromonas (Genbank accession: YP_001143607), alpha-toxin fromClostridium septicum (ABD65254), ε-toxin form Clostridium perfringens(CAA43104), parasporin-2 (PS2) from Bacillus thuringiensis (BAC79010).In addition, hydralysins from Cnidaria (XP_002158854), hemolytic lectinfrom the parasitic mushroom Laetiporus sulphureus (BAC78489), andenterolobin produced by the seeds of the Brazilian tree (P81007).

Structural modeling revealed that MP467 (SEQ ID NO: 2) has a significantstructural similarity to a well-studied aerolysin family. The structureof MP467 (SEQ ID NO: 2) was modeled using standard homology modelingtechniques available within the Discovery Studio 3.5° software(Copyright 2005-12 Accelrys Software). In brief, the MP467 amino acidsequence (SEQ ID NO: 2) was used as the query sequence to BLAST againstthe available structures in the Protein Databank (PBD). The top scoringBLAST hit was parasporin-2 from Bacillus thuringiensis (PDB ID 2ZTB)with an overall sequence identity of 42% covering 245 amino acids of thequery sequence and an E-value of 4.0674e-45. The parasporin-2 structure(2ztb) was used as the structural template for the MP467 sequence (SEQID NO: 2). During the homology modeling procedure 20 models for MP467(SEQ ID NO: 2) were generated, energy minimized with high optimizationsetting (Discovery Studio 3.5°) and scored. The lowest energy model wasused in the following structural analysis of MP467 (SEQ ID NO: 2).

The overall structure of modeled MP467 (SEQ ID NO: 2) shares a highdegree of similarity to the parasporin-2 structure (2ztb) the largestdifferences resulting from a two residue deletion in the loop connecting1311 and 1312 (Akiba, 2009) Briefly the model for MP467 (SEQ ID NO: 2)is comprised of an elongated β-strand structure (approximately 113 Å×18Å×25 Å) aligned with its long axis, similar to aerolysin-typeβ-pore-forming toxins (Szczesny et al., 2011). According to theaerolysin structure convention, the PS2 structure (2ztb) consists ofthree structural domains with the two longest beta strands (134 and 135)running through the entire three-domains (FIGS. 7 and 8). In hemolyticlectin from Laetiporus sulphureus, β4 and β5 are short and only coverthe domain 2 and 3 while they have a large subdomain extensionencompassing half of the receptor binding domain in aerolysin. In orderto better refine the structure core and reveal more structuralhomologues, the variable elements (residue 52-116 and 240-274, domain I)were removed from PS2 structure (2ztb) and structural similarity wasdetermined using the Dali algorithm (Holm L, Rosenström P (2010) Daliserver: conservation mapping in 3D. Nucl. Acids Res. 38, W545-549). Thecore domain is similar to ε-toxin (PDB:1uyj, Cole et al. 2004),hemolytic lectin from mushroom (1w3a, 1w3f, and 1w3g, Mancheño et a;2005), aerolysin from various sources (3g4o, 1pre, etc., Rossjohn etal., 1998), and enterotoxin (2xh6 and amx2, Kitadokoro et al., 2011),consistent with the sequence comparison. Although these toxins havedrastically different Domain I structures likely reflecting theirdistinct target specificity, they exhibit the same general topology inDomain II and Domain III establishing their evolutionary and functionalrelevancy.

Domain I of MP467 (SEQ ID NO: 2) is comprised of residues 1 to 65, andresidues 187 to 223 (Table 3, FIG. 9). The structure of domain Iconsists of an anti-parallel β-sheet with four short strands in betweenhelix 2 and 3. The β-turn between strand 11 and strand 12 is shortenedby two residues in the MP467 model compared with parasporin-2. Domain Iof MP467 (SEQ ID NO: 2) contains a surface hydrophobic patch consistingof Phe26, Val48, Pro50, Ile52, Tyr56, Met193, Val202, His205, Tyr206,Phe207, Trp208, Phe209, and Leu210. The hydrophobic patch (FIG. 9) isapproximately 198 Å² (17.7 Å×11.2 Å) encompassing strand 12, helix 2,helix 3 and the type 1′ β-turn between strand 11 and strand 12 (motifshown in SEQ ID NO: 13). Literature evidence strongly suggests thatDomain I of the aerolysin-type beta-pore-forming-toxins (β-PFTs) isinvolved in receptor binding (Abe, 2008) (Tateno, 2003) (Lafont, 2004)(Song, 1996) (Olson, 1999).

Domain II and III adopt a highly twisted topology made of almostentirely β strands and defines as the conserved aerolysin fold (Szczesnyet al., 2011). Domain II is a five-stranded anti-parallel β-sheet(β5/β6-β11-β13-β7-β10, where both β5 and β6 can be viewed as a brokenone long strand) patched on one side by an amphipathic β-hairpin β8 andβ9 (FIG. 9). This hairpin stemmed from β7 and β10 forms a hydrophobiccore with the central β-sheet, but it is too thin to cover the wholeinner surface. This has forced the edge of central sheet to curl towardthe hairpin and to wrap the uncovered surface. In enterotoxin CPE(3am2), the corresponding β-hairpin undergoes a drastic conformationalchange and assumes an α-helix, but its amphipathic characteristicessential for structural stability is well preserved. The same fiveβ-strands of domain 2 extend and refold into domain 3 with abeta-sandwich structure. Three strands β5/6-β11-β13 make a 180° twist inmiddle forming a new 3 stranded β-sheet as one side of β-sandwich. Theβ7 and β10 spray from the central sheet, also twist in middle, andhydrophobically pack against strands β5/β6-β11-β13. These naturalβ-sheet twisting points provide a convenient domain divider. The β5/β6usually does not hold the β conformation along its whole length due totwist and separates into two linked β strands as β5 and β6. Apparently,this usual topology strikes a balance between protein structuralstability and flexibility critical to conformational transition fromsoluble to pore forming state.

On the surface of domain II and III, there is a stripe of solventexposed serine and threonine residues (FIG. 7). β-strand 10 contains 3serine and 4 threonine residues with average solvent accessibility of78.5 Å². These residues occur in an “every-other-one” motif from N toC-terminus of the β-strand 10. β-strand 7 also contains a stretch of 3serine and 2 threonine residues with average solvent exposure of 59.3Å². This distinct serine/threonine stripe has been observed in theaerolysin-type beta-pore-forming-toxins (β-PFTs). (Rossjohn, 1998) Ithas been proposed that this feature of parasporin-2 may be involved inaligning the molecule parallel to membrane after initial receptorbinding by domain I. (Akiba, 2009)

Based on structural comparison and biochemistry essay, variousaerolysin-like toxins despite their distinct target specificity arethought to share the same mode of action, β-barrel pore formation inmembrane. The toxin is produced as a soluble protein which diffusestowards its target cell where it binds via specific surface receptors.Once receptor bound, the toxin undergoes circular polymerization,generating ring like structures that subsequently insert into themembrane and form a pore. While aerolysin and ε-toxin form heptamers,the stoichiometry might differ between members. Multiple lines ofevidences back up a notation that the pore forming is carried out withthe conserved beta hairpin. First, the β-barrel conformation acrossmembrane positions residues along the pore wall facing eitherhydrophobic lipid bilayer or hydrophilic pore lumen. Thus, the sequenceof the transmembrane insertion elements must at least have thealternating pattern of polar and hydrophobic residues although anydistinct sequence conservation might be not strictly required. Thesequence alignment among the inserting β-hairpin from the typicalaerolysin-like toxins including MP467 demonstrated that this amphipathicpattern is largely persevered (FIG. 5). In fact, the hairpin is one ofmost conserved elements on the whole sequence. Second, the crystalstructure of heptameric pore of Staphylococcus aureus α-hemolysinpresented a membrane β pore confirmation (FIG. 6), and clearly showedthat the pore stem is made of amphipathic β-hairpins (Song et al.,1996). Despite the sequence difference from aerolysin, the hairpinalternating pattern is also observed (FIG. 5). Third, it has beenexperimentally demonstrated that a similar amphipathic β-hairpin is thepore-forming element in Clostridium septicum alpha toxin (Melton et al.,2009), a sequence homolog of aerolysin and MP467. Using deletionmutagenesis, cysteine-scanning mutagenesis and multiplespectrofluorimetric methods, the applicants showed that either removingthe hairpin by deletion or restricting its movement by engineereddisulfide abolishes alpha toxin's pore forming capability but does notaffect other functions.

TABLE 3 Domain residues of MP467 (SEQ ID NO: 2) Domain Amino acidposition ranges I 1-65; 187-223 II 66-79; 104-154; 175-186; 224-234 III80-103; 155-174; 235-246

The constellation of hydrophobic residues within the hydrophobic patchand the conformation of the type 1′ β-turn in domain I and thearrangement of serine/threonine residues throughout domain II and IIIare believed to be in part responsible for the insecticidal activity ofMP467 (SEQ ID NO: 2). Through sequence and structural comparisons it ispredict that the PS2 (Cry46A) and hydralysin proteins should havesimilar activity to MP467 (SEQ ID NO: 2).

Example 2: Lepidoptera and Coleoptera Assays with Purified Proteins

Insecticidal activity bioassay screens were conducted to evaluate theeffects of the insecticidal proteins on a variety of Lepidopteraspecies: European corn borer (Ostrinia nubilalis), corn earworm(Helicoverpa zea), black cutworm (Agrotis ipsilon), fall armyworm(Spodoptera frugiperda), Soybean looper (Pseudoplusia includens) andVelvet bean caterpillar (Anticarsia gemmatalis), and a Coleoptera specie(Western corn rootworm (Diabrotica virgifera).

Lepidoptera feeding assays were conducted on an artificial dietcontaining the cleared lysates of bacterial strains in a 96 well plateset up. The cleared lysate was incorporated with theLepidopteran-specific artificial diet in a ratio of 20 ul cleared lysateand 40 ul of diet mixture. Two to five neonate larvas were placed ineach well to feed ad libitum for 5 days. Results were expressed aspositive for larvae reactions such as stunting and or mortality. Resultswere expressed as negative if the larvae were similar to the negativecontrol that is feeding diet to which the above buffer only has beenapplied. Each cleared lysate was assayed on European corn borer(Ostrinia nubilalis), corn earworm (Helicoverpa zea), black cutworm(Agrotis ipsilon), fall armyworm (Spodoptera frugiperda), Soybean looper(Pseudoplusia includens) and Velvet bean caterpillar (Anticarsiagemmatalis). A series of concentrations of the purified protein samplewas assayed against those insects and concentrations for 50% mortality(LC50) or inhibition of 50% of the individuals (IC50) were calculated.The results are shown in Tables 4 and 5.

Coleoptera feeding assays were conducted on an artificial dietcontaining the cleared lysates of bacterial strains in a 96 well plateset up. The cleared lysate was incorporated with thecoleopteran-specific artificial diet in a ratio of 10 ul cleared lysateand 50 ul of diet mixture. Two to five Western corn rootworm (Diabroticavirgifera) neonate larva were placed in each well to feed ad libitum for5 days. Results were expressed as positive for larvae reactions such asstunting and or mortality. Results were expressed as negative if thelarvae were similar to the negative control that is feeding diet towhich the above buffer only has been applied. For MP467 (SEQ ID NO: 2) aseries of concentrations of the purified protein sample was assayedagainst those insects and concentrations for 50% mortality (LC50) orinhibition of 50% of the individuals (IC50) were calculated. Theinsecticidal assay results for MP467 (SEQ ID NO: 2) is shown in Table 4.

TABLE 4 Insecticidal activity of MP467 (SEQ ID NO: 2) Lower 95% Upper95% Insect LC/IC MP467, ppm Confidence Limit Confidence Limit CEW LC50279.8 61.71 1268 IC50 146.3 121.9 177.6 FAW LC50 >320 (40% mort.) IC50295.6 236.3 443.3 BCW LC50 204.8 173.4 244.2 IC50 111.9 96.28 130.7 SBLLC50 212.5 177 252.3 IC50 120.18 99.5 142.4 VBC LC50 54.72 41.25 67.1IC50 43.49 32.42 53.3 Coleopteran WCRW LC50 65.84 56.07 76 IC50 25.9622.94 29.61 Hemipteran Lygus LC50 22.2 19.6 25.2

For Cry46Aa (SEQ ID NO: 10) the response of insects towards the proteinswas scored using a 0-3 numerical scoring system based on the size andmortality of the larvae in each well. If no response (or normal growth)was seen, a score of 0 was given. When the growth was slightly retarded,a score of 1 was given. A score of 2 meant that the larvae were severelyretarded in growth (close to neonate size). A score of 3 meant death toall the larvae in the well. The percent response (% Response) for eachtreatment was calculated by dividing the total score, a sum of scoresfrom replicated wells for each treatment, by the total highest possiblescores and multiplying by 100 to yield “% Response”. For example, if onetreatment (one sample, one dose) had 6 replicated wells, the totalhighest possible score would be 3×6=18. An observed set of scores of 3,2, 2, 3, 2, 2 for six wells at a given dose for a given variant wouldresult in (14/18)×100=78% Response. Therefore this scoring system was acombination of feeding inhibition (I) and lethality (L). ILC50(concentration for 50% response) was calculated by using Probitanalysis. The insecticidal activity for Cry46Aa (SEQ ID NO: 10) is shownin Table 5.

TABLE 5 Insecticidal activity of Cry46Aa (SEQ ID NO: 10) Insect ILC50,ppm WCRW 32 Lygus 69 ECB >230 BCW 32 CEW 195 FAW >230 SBL 36

Example 3: Lygus Bioassay with Purified Proteins

Lygus (Lygus hesperus) bioassays were conducted using the cell lysatesamples mixed with insect diet (Bio-Sery F9644B) in each well of a 96well bioassay plate (BD Falcon™ 353910). A variable number of Lygushesperus second instar nymphs (2 to 7) were placed into each well of a96 well plate. The assay was run four days at 25° C. and then was scoredfor insect mortality and stunting of insect growth. A series ofconcentrations of the purified protein sample was assayed against thoseinsects and concentrations for 50% mortality (LC50) or inhibition of 50%of the individuals (ILC50) were calculated.

The Lygus assay was run for 4 days with 15 2nd stage instars per petridish with 3 reps per dose. Doses were 40 ul sample at 3 mg/ml MP467 (SEQID NO: 2) or Cry46Aa (SEQ ID NO: 10)+360 ul diet. The results are inTable 4 and Table 5.

Example 4: Identification of Motifs for Insecticidal Activity: Domain IHydrophobic Patch Mutagenesis

Saturation mutagenesis was performed on residues in the hydrophobicpatch of domain I, specifically Tyr206 and Trp208 as well as sitedirected mutagenesis at Phe207 and Phe209. These residues appear to beimportant in determining hemolytic and insecticidal activities of MP467.Results for Western Corn Rootworm (WCRVV) are shown in Table 6.

In Table 6, “R” denotes a reduction in activity over the wild-type, “=”denotes the activity similar to wild-type and “-” denotes no WCRWactivity.

TABLE 6 AA position and Gene Name change WCRW Bioassay Data MP467-M1W208A R MP467-M2 D204A = MP467-M3 H205A R MP467-M4 Y206A R MP467-M5F207A R MP467-M6 F209A R MP467-M7 Y56A R MP467-M8 H58A R MP467-M9 W208RR MP467-M10 W208N R MP467-M11 W208D R MP467-M12 W208C R MP467-M13 W208Q= MP467-M14 W208E = MP467-M15 W208G — MP467-M16 W208H = MP467-M17 W208I= MP467-M18 W208L = MP467-M19 W208K R MP467-M20 W208M = MP467-M21 W208F= MP467-M22 W208P — MP467-M23 W208S = MP467-M24 W208T = MP467-M25 W208Y= MP467-M26 W208V = MP467-M40 Y206R, W208M — MP467-M41 Y206N, W208M RMP467-M42 Y206D, W208M — MP467-M43 Y206C, W208M — MP467-M44 Y206Q, W208M— MP467-M45 Y206E, W208M — MP467-M46 Y206G, W208M — MP467-M47 Y206H,W208M — MP467-M48 Y206I, W208M — MP467-M49 Y206L, W208M — MP467-M50Y206K, W208M — MP467-M51 Y206M, W208M — MP467-M52 Y206F, W208M =MP467-M53 Y206S, W208M — MP467-M54 Y206T, W208M — MP467-M55 Y206W, W208MR MP467-M56 Y206V, W208M — MP467-M57 Y206R R MP467-M58 Y206N = MP467-M59Y206D R MP467-M60 Y206C — MP467-M61 Y206Q R MP467-M62 Y206E — MP467-M63Y206G — MP467-M64 Y206H = MP467-M65 Y206I = MP467-M66 Y206L = MP467-M67Y206K — MP467-M68 Y206M = MP467-M69 Y206F R MP467-M70 Y206S R MP467-M71Y206T R MP467-M72 Y206W = MP467-M73 Y206V R

Example 5. MP467 Variants with Reduced Hemolytic Activity

Cry46Aa (parasporin SEQ ID NO: 10) has been reported in the literatureto have hemolytic activity. Similarly, MP467 (SEQ ID NO: 2) in additionto insecticidal activity showed hemolytic activity. Selected MP467hydrophobic patch variants shown in Table 6 (Example 4) having aminoacid substitutions at positions 56, 58, 204, 205, 206, 207, 208 and 209of SEQ ID NO: 2 were assayed for hemolytic activity.

The Cry46Aa (parasporin-2Aa SEQ ID NO: 10), MP467 (SEQ ID NO: 2), andthe MP467 hydrophobic patch variants (Table 6) were assayed forhemolytic activity essentially as described by Bernheimer, A. W. (Assayof hemolytic toxins. Methods Enzymol. 165: 213-217, 1988); and Rowe, G.E. and R. A. Welch (Assays of hemolytic toxins. Methods Enzymol.235:657-667, 1994). Briefly, human red blood cells (Rockland Antibodiesand Assays Lot#BP9525D) were washed with PBS (50 mM phosphate buffer, pH7.4, 150 mM NaCl) and centrifuged at 400×g for 5 min. The washes wererepeated until the supernatant was visibly clear of hemoglobin. Theerythrocyte suspension was adjusted to 1% with PBS supplemented with0.1% bovine serum albumin and 0.2 ml of the 1% erythrocyte suspensionwas incubated at 37° C. with 0.2 ml tested protein/extract in PBS. Theincubated erythrocyte suspension was centrifuge at 120×g for 7 min toremove undamaged RBCs. The resulting supernatant (0.3 ml) wastransferred into each well of 96-well plate. The concentration ofreleased hemoglobin was determined by reading absorbance at 545 nm in aspectrophotometer against a control background (0.5 ml erythrocytesuspension with 0.5 ml PBS). The 100% hemolysis standard was determinedby incubating 0.2 ml of the 1% erythrocyte suspension at 37° C. with 0.2ml of 0.1% (final) Triton ×100 in PBS. The hemolytic results are shownin Table 7.

TABLE 7 Variant AA Gene Name sequence Hemolysis (+/−) % HemolysisMP467-M1 W208A Negative MP467-M2 D204A +(strong) 99.3 MP467-M3 H205A+(strong) 98.8 MP467-M4 Y206A Negative MP467-M5 F207A Negative MP467-M6F209A Negative MP467-M7 Y56A Negative MP467-M8 H58A +(strong) 100MP467-M9 W208R Negative MP467-M10 W208N Negative MP467-M11 W208DNegative MP467-M12 W208C Negative MP467-M13 W208Q Negative MP467-M14W208E Negative MP467-M15 W208G Negative MP467-M16 W208H NegativeMP467-M17 W208I +(medium) 62.5 MP467-M18 W208L Negative MP467-M19 W208KNegative MP467-M20 W208M Negative MP467-M21 W208F +(medium) 43.6MP467-M22 W208P Negative MP467-M23 W208S Negative MP467-M24 W208TNegative MP467-M25 W208Y +(strong) 87 MP467-M26 W208V Negative MP467(SEQ DHYFWFL (amino +(strong) 99.8 ID NO: 2) acids 204 to 210 of SEQ IDNO: 2)

Example 6: Transient Expression and Insect Bioassay on Transient LeafTissues

The polynucleotide of SEQ ID NO: 28 (MP467 MODA), with maize optimizedcodons, encoding MP467 (SEQ ID NO: 2) was cloned into a transientexpression vector under control of the maize ubiquitin promoter(Christensen and Quail, (1996) Transgenic Research 5:213-218) and aduplicated version of the promoter from the mirabilis mosaic virus (DMMVPRO; Dey and Maiti, (1999) Plant Mol. Biol., 40:771-82). Theagro-infiltration method of introducing an Agrobacterium cell suspensionto plant cells of intact tissues so that reproducible infection andsubsequent plant derived transgene expression may be measured or studiedis well known in the art (Kapila, et. al., (1997) Plant Science122:101-108). Briefly, young plantlets of maize were agro-infiltratedwith normalized bacterial cell cultures of test and control strains.Leaf discs were generated from each plantlet and infested WCRW(Diabrotica virgifera) along with appropriate controls. The degree ofconsumption of green leaf tissues was scored after 2 days ofinfestation. Purified MP467 protein (SEQ ID NO: 2) was used as astandard.

After 2d feeding, the amount of leaf tissue consumed by WCRW larvae wasscored across 24 disks per treatment. When compare to negative control(DsRed), tissue accumulating MP467 was significantly less damaged acrossthe 24 disks. A particular promoter/gene design combination (DMMVPRO:MP467 (MODA), provided as much protection as a known positivecontrol.

Example 7: Identification of MP467 Homologs

The amino acid sequence of MP467 (SEQ ID NO: 2) was BLAST searched(Basic Local Alignment Search Tool; Altschul, et al., (1993) J. Mol.Biol. 215:403-410; see also ncbi.nlm.nih.gov/BLAST/, which can beaccessed using the www prefix) against public and proprietaryDUPONT-PIONEER internal databases that included insecticidal polypeptidesequences. The search identified a MP467 homolog, MP812 (SEQ ID NO: 24)from a Bacillus thuringiensis strain designated as JH50823-1. WhileMP812 (SEQ ID NO: 24) has only 19% sequence identity and 38.3% sequencesimilarity to MP467 (SEQ ID NO: 2) the overall three-domain insecticidalstructure, the surface hydrophobic patch and type 1′ β-turn in Domain I,and the stripe of solvent exposed serine and threonine residues on thesurface of Domain II and Domain III are conserved. The percent identityand similarity between the amino acid sequences of MP467 (SEQ ID NO: 2),MP812 (SEQ ID NO: 24), and Cry46Ab (SEQ ID NO: 12) are shown in Table 6and Table 7 respectively.

TABLE 6 Cry46Ab MP812 (SEQ ID NO: 12) (SEQ ID NO: 24) MP467 (SEQ ID NO:2) 37.2% 19.0% Cry46Ab (SEQ ID NO: 12) — 23.1% MP812 (SEQ ID NO: 24) — —

TABLE 7 Cry46Ab (SEQ ID NO: 12) MP812 (SEQ ID NO: 24) MP467 (SEQ ID NO:2) 52.6% 38.3% Cry46Ab (SEQ ID NO: 12) — 40.0% MP812 (SEQ ID NO: 24) — —

In the primary screen against European corn borer (Ostrinia nubilalis),corn earworm (Helicoverpa zea), black cutworm (Agrotis ipsilon), fallarmyworm (Spodoptera frugiperda), Soybean looper (Pseudoplusiaincludens) and Velvet bean caterpillar (Anticarsia gemmatalis), andWestern corn rootworm (Diabrotica virgifera) MP812 (SEQ ID NO: 24)showed insecticidal activity against Western corn rootworm (Diabroticavirgifera) at a concentration of 20 ug/cm². The assays were repeated forWestern corn rootworm (Diabrotica virgifera) and corn earworm(Helicoverpa zea) side by side for MP467 (SEQ ID NO: 2) and MP812 (SEQID NO: 24) from 75 ug/cm² to 4.69 ug/cm². The response of insectstowards the proteins was scored using a 0-3 numerical scoring systembased on the size and mortality of the larvae in each well. If noresponse (or normal growth) was seen, a score of 0 was given. When thegrowth was slightly retarded, a score of 1 was given. A score of 2 meantthat the larvae were severely retarded in growth (close to neonatesize). A score of 3 meant death to all the larvae in the well. Theresults are shown in Table 8.

TABLE 8 Sample Conc. Rep1 Rep2 Rep3 Pep4 Avg. WCRW MP467 75 ug/cm² 3 3 23 2.75 MP467 37.5 ug/cm² 2 2 2 2 2 MP467 18.75 ug/cm² 2 2 2 2 2 MP4679.375 ug/cm² 1 0 0 1 0.5 MP467 4.69 ug/cm² 0 0 0 0 0 MP467 0 0 0 0 0 0MP812 75 ug/cm² 2 2 3 3 2.5 MP812 37.5 ug/cm² 2 2 2 2 2 MP812 18.75ug/cm² 2 2 2 2 2 MP812 9.375 ug/cm² 0 0 0 0 0 MP812 4.69 ug/cm² 0 0 0 00 MP812 0 0 0 0 0 0 CEW MP467 75 ug/cm² 2 2 2 2 2 MP467 37.5 ug/cm² 2 12 2 1.75 MP467 18.75 ug/cm² 1 1 1 0 0.75 MP467 9.375 ug/cm² 0 0 0 0 0MP467 4.69 ug/cm² 0 0 0 0 0 MP467 0 0 0 0 0 0 MP812 75 ug/cm² 0 0 0 0 0MP812 37.5 ug/cm² 0 0 0 0 0 MP812 18.75 ug/cm² 0 0 0 0 0 MP812 9.375ug/cm² 0 0 0 0 0 MP812 4.69 ug/cm² 0 0 0 0 0 MP812 0 0 0 0 0 0

Example 8: Southern Green Stinkbug Assay with Purified Protein

Southern green stinkbug (Nezara viridula) eggs were collected from alaboratory maintained colony and kept in an incubator at 27° C. with 65%relative humidity. After hatching, the insects were allowed to feed ongreen beans with or without the addition of green peas. Thereafter,freshly molted second instar stinkbugs were transferred onto a modifiedartificial Lygus diet (Bioserve; Lygus Hesperus diet, catalog # F9644B)supplemented either with MP467 (SEQ ID NO: 2) or MP812 (SEQ ID NO: 24)or water (as control). Five second instar stinkbugs per bioassay werefed with varying dosages of insecticidal proteins supplemented in theartificial diet. The diet with insecticidal proteins or water waschanged every two days and the bioassay observations on stunting and/ormortality were taken on day 7. Mortality was recorded at the conclusionof the assay. In addition, remaining insects were weighed to recordstunting at the conclusion of the assay. The results are shown in Table9.

TABLE 9 Weight of Individual # 2^(nd) 3^(rd) % 3^(rd) bugs bug # SampleRemaining instar instar instar remaining Average Dead H2O 5 0 5 10046.53 9.31 0 H2O 5 0 5 100 53.12 10.62 0 H2O 5 0 5 100 48.56 9.71 0 H2O5 0 5 100 62.88 12.58 0 H2O 4 1 3 75 40.48 8.1 1 H2O 5 0 5 100 59.6711.93 0 MP467 5 0 5 100 58.61 11.72 0  25 ppm MP467 5 1 4 80 38.52 7.7 0 50 ppm MP467 3 3 0 0 9.5 1.9 2 100 ppm MP467 3 3 0 0 6.72 1.34 2 200ppm MP467 0 0 0 0 0 0 5 400 ppm MP812 2 0 2 100 25.2 5.04 3  25 ppmMP812 4 0 4 100 29.73 5.95 1  50 ppm MP812 3 0 3 100 17.81 3.56 2 100ppm MP812 4 2 2 50 16.37 3.27 1 150 ppm MP812 3 2 1 33.33 9.22 1.84 2200 ppm MP812 300 ppm 0 0 0 0 0 0 5

Example 9: Transformation of Maize by Particle Bombardment andRegeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aDNA molecule containing the toxin nucleotide sequence (e.g., SEQ ID NOs:1 or 9) operably linked to a suitable promoter and a suitable selectablemarker gene (e.g. PAT, Wohlleben, et al., (1988) Gene 70: 25-37; whichconfers resistance to the herbicide Bialaphos). Alternatively, theselectable marker gene is provided on a separate DNA molecule.Transformation is performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% CLOROX™ bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5 cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising a nucleotide sequence (e.g., SEQ ID NO: 1)operably linked to an ubiquitin promoter is constructed. For example, asuitable transformation vector comprises a UBI1 promoter from Zea mays,a 5′ UTR from UBI1 and a UBI1 intron, in combination with a PinIIterminator. The vector additionally contains a PAT selectable markergene driven by a CAMV35S promoter and includes a CAMV35S terminator.Optionally, the selectable marker can reside on a separate plasmid. ADNA molecule comprising a toxin nucleotide sequence as well as a PATselectable marker is precipitated onto 1.1 μm (average diameter)tungsten pellets using a CaCl₂ precipitation procedure as follows:

-   -   100 μL prepared tungsten particles in water    -   10 μL (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)    -   100 μL 2.5 M CaCl₂    -   10 μL 0.1 M spermidine

Each reagent is added sequentially to a tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 mL 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μL 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μLspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the toxin by assaysknown in the art or as described above.

Bombardment and Culture Media

Bombardment medium (560Y) comprises 4.0 g/L N6 basal salts (SIGMAC-1416), 1.0 mL/L Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/Lthiamine HCl, 120.0 g/L sucrose, 1.0 mg/L 2,4-D and 2.88 g/L L-proline(brought to volume with deionized H₂O following adjustment to pH 5.8with KOH); 2.0 g/L Gelrite™ (added after bringing to volume with dlH₂O); and 8.5 mg/L silver nitrate (added after sterilizing the mediumand cooling to room temperature). Selection medium (560R) comprises 4.0g/L N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vitamin Mix(1000× SIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L sucrose, and 2.0mg/L 2,4-D (brought to volume with dl H₂O following adjustment to pH 5.8with KOH); 3.0 g/L Gelrite™ (added after bringing to volume with dlH₂O); and 0.85 mg/L silver nitrate and 3.0 mg/L Bialaphos (both addedafter sterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/L MS salts (GIBCO11117-074), 5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/L thiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog (1962)Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 mg/L zeatin, 60 g/Lsucrose, and 1.0 mL/L of 0.1 mM abscisic acid (brought to volume withpolished dl H₂O after adjusting to pH 5.6); 3.0 g/L Gelrite™ (addedafter bringing to volume with dl H₂O); and 1.0 mg/L indoleacetic acidand 3.0 mg/L Bialaphos (added after sterilizing the medium and coolingto 60 C).

Hormone-free medium (272V) comprises 4.3 g/L MS salts (GIBCO 11117-074),5.0 mL/L MS vitamins stock solution (0.100 g/L nicotinic acid, 0.02 g/Lthiamine HCl, 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought tovolume with polished dl H₂O), 0.1 g/L myo-inositol, and 40.0 g/L sucrose(brought to volume with polished dl H₂O after adjusting pH to 5.6); and6 g/L Bacto-agar (added after bringing to volume with polished dl H₂O),sterilized and cooled to 60° C.

Example 10: Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with a toxinnucleotide sequence (e.g., SEQ ID NO: 1 or 9), the method of Zhao can beused (U.S. Pat. No. 5,981,840 and PCT patent publication WO98/32326; thecontents of which are hereby incorporated by reference). Briefly,immature embryos are isolated from maize and the embryos contacted witha suspension of Agrobacterium under conditions whereby the bacteria arecapable of transferring the toxin nucleotide sequence (SEQ ID NO: 1) toat least one cell of at least one of the immature embryos (step 1: theinfection step). In this step the immature embryos can be immersed in anAgrobacterium suspension for the initiation of inoculation. The embryosare co-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos can be cultured on solidmedium following the infection step. Following this co-cultivationperiod an optional “resting” step is contemplated. In this resting step,the embryos are incubated in the presence of at least one antibioticknown to inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos can be cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos arecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryosare cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium can be cultured on solid medium to regenerate theplants.

Example 11: Transformation of Soybean Embryos

Soybean embryos are bombarded with a plasmid containing the toxinnucleotide sequence of SEQ ID NO: 1 operably linked to a suitablepromoter as follows. To induce somatic embryos, cotyledons, 3-5 mm inlength dissected from surface-sterilized, immature seeds of anappropriate soybean cultivar are cultured in the light or dark at 26° C.on an appropriate agar medium for six to ten weeks. Somatic embryosproducing secondary embryos are then excised and placed into a suitableliquid medium. After repeated selection for clusters of somatic embryosthat multiplied as early, globular-staged embryos, the suspensions aremaintained as described below.

Soybean embryogenic suspension cultures can be maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 mL ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation includes, but is not limited to: the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188), and the 3′ region of thenopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising a toxin nucleotidesequence (e.g., SEQ ID NO: 1) operably linked to a suitable promoter canbe isolated as a restriction fragment. This fragment can then beinserted into a unique restriction site of the vector carrying themarker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1M), and 50 μL CaCl₂(2.5M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this disclosure pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

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That which is claimed:
 1. A polynucleotide comprising a heteroloqousregulatory element operably linked to an isolated nucleic acid moleculeencoding a three-domain insecticidal protein comprising a polypeptidesequence having at least 80% sequence identity to SEQ ID NO: 2 and thefollowing protein structure is present in the three-domain insecticidalprotein: a) a Domain I, comprising i) a surface hydrophobic patchcomprising residues corresponding to Phe26, Val48, Pro50, Ile52, Tyr56,Met193, Val202, His205, Tyr206, Phe207, Trp208, Phe209, and Leu210 ofSEQ ID NO: 2; ii) an anti-parallel β-sheet with four short strands andfour α-helices designated as helix 1, helix 2, helix 3 and helix 4; andiii) a type 1′ β-turn comprising an amino acid sequence motif asrepresented by SEQ ID NO: 25; b) a Domain II; and c) a Domain III,wherein the surface of Domain II and Domain III comprises a stripe ofsolvent exposed serine and threonine residues.
 2. The isolated nucleicacid molecule of claim 1 encoding the three-domain insecticidal protein,wherein the anti-parallel β-sheet of Domain I of the protein structureof the three-domain insecticidal protein further comprises theC-terminal end of strand 11, strand 12, and the type 1′ β-turn joinsstrand 11 and strand
 12. 3. The isolated nucleic acid molecule of claim1 encoding the three-domain insecticidal protein, wherein Domain I ofthe protein structure of the three-domain insecticidal protein furthercomprises a β-hairpin between helix 2 and helix
 3. 4. The isolatednucleic acid molecule of claim 1 encoding the three-domain insecticidalprotein, wherein the surface hydrophobic patch of Domain I of theprotein structure of the three-domain insecticidal protein comprises theresidues from strand 12, helix 2, helix 3, and the type 1′ β-turn ofDomain I of the protein structure of the three-domain insecticidalprotein comprises the residues between strand 11 and strand
 12. 5. Theisolated nucleic acid molecule of claim 1 encoding a three-domaininsecticidal protein, wherein the surface hydrophobic patch of Domain Iof the protein structure of the three-domain insecticidal protein isabout 180-220 Å².
 6. The isolated nucleic acid molecule of claim 1encoding a three-domain insecticidal protein, wherein Domain II of theprotein structure of the three-domain insecticidal protein comprises afive-stranded anti-parallel β-sheet comprising β-strandsβ5/β6-β11-β13-β7-β10 patched on one side by an amphipathic β-hairpinstemmed from β2 and β10.
 7. The isolated nucleic acid molecule of claim1 encoding a three-domain insecticidal protein, wherein the stripe ofexposed serine and threonine residues on the surface of Domain II andDomain III of the protein structure of the three-domain insecticidalprotein comprises: 3 serine and 4 threonine residues in an alternatingmotif on strand 11, and 3 serine and 2 threonine residues on strand 7.8. A DNA construct comprising the nucleic acid molecule of claim 1, 2,3, 4, 5, 6 or
 7. 9. A transgenic plant comprising the DNA construct ofclaim
 8. 10. A method for protecting a plant from a pest, comprisingintroducing into said plant or cell thereof at least one DNA constructof claim 8.